SUTTER INSTRUMENT P-1000 User Manual

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P----1000

Flaming/Brown Micropipette

Flaming/Brown MicropipetteFlaming/Brown Micropipette

Puller System

Puller SystemPuller System

Operation Manual

Operation ManualOperation Manual

1000

10001000

Rev. 1.06 ( 20110211)

One Digital Drive

Novato, CA 94949

Voice: 415-883-0128 Web: www.sutter.com Fax: 415-883-0572 Email: info@sutter.com



Thermolock™ is a trademark of Sutter Instrument Company.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

iii

DIS C LAIM ER

DIS C LAIM ERDIS C LAIM ER

The pipette puller Model P-1000 is designed for the specific use of creating micropipettes and no other use is recommended.

This instrument creates items that should only be used in a laboratory environment for use on animal tissues. It is not intended for use, nor should be used, in human experimentation, or applied to humans in any way. This is not a medical device.

Do not open or attempt to repair the instrument without expressed and explicit instructions from Sutter Instrument Company. Extreme heat and high voltages are present and could cause injury.

Do not allow unauthorized and or untrained operatives to use this device.

Any misuse will be the sole responsibility of the user/owner and Sutter Instruments assumes no implied or inferred liability for direct or consequential damages from this instrument if it is operated or used in any way other than for which it is designed.

SAF E TY W ARNI N GS A ND P RECA UTIO N S

SAF E TY W ARNI N GS A ND P RECA UTIO N SSAF E TY W ARNI N GS A ND P RECA UTIO N S

Main Fuse

Main FuseMain Fuse



Avoiding Electrical Shock or Fire Injury

Avoiding Electrical Shock or Fire InjuryAvoiding Electrical Shock or Fire Injury

 



Back Injury Prevention

Back Injury PreventionBack Injury Prevention

Replace

Replace fuse only with the same type and rating:

Replace Replace

Type:

Type: 5 x 20 mm glass tube, Medium Time Delay (IEC

Type:Type:

Rating:

Rating: T4A 250V (Time Delay, 4 Amps, 250 Volts)

Rating:Rating:

Examples:

Examples: Bussmann S506

Examples:Examples:

A spare fuse is provided, located in the power input module. Please refer to

A spare fuse is provided, located in the power input module. Please refer to Appendix C

A spare fuse is provided, located in the power input module. Please refer to A spare fuse is provided, located in the power input module. Please refer to for more details on fuse ratings and for instructions on how to change the fuse.

for more details on fuse ratings and for instructions on how to change the fuse.

for more details on fuse ratings and for instructions on how to change the fuse.for more details on fuse ratings and for instructions on how to change the fuse.

To prevent fire or shock hazard do not expose the unit to rain or moisture.

To prevent fire or shock hazard do not expose the unit to rain or moisture.To prevent fire or shock hazard do not expose the unit to rain or moisture.

Use only a properly grounded

Use only a properly grounded power source; appropriately rated for use with this

Use only a properly grounded Use only a properly grounded instrument. Always use the grounded power supply cord set provided to connect the unit

instrument. Always use the grounded power supply cord set provided to connect the unit

instrument. Always use the grounded power supply cord set provided to connect the unit instrument. Always use the grounded power supply cord set provided to connect the unit to the grounded outlet (3

to the grounded outlet (3----prong). This is required to protect you from injury in the event

to the grounded outlet (3to the grounded outlet (3 that an electrical hazard

that an electrical hazard occurs.

that an electrical hazard that an electrical hazard

Do not disassemble the unit. Refer servicing to Sutter Instrument Company.

Do not disassemble the unit. Refer servicing to Sutter Instrument Company.Do not disassemble the unit. Refer servicing to Sutter Instrument Company.

fuse only with the same type and rating:

fuse only with the same type and rating: fuse only with the same type and rating:

5 x 20 mm glass tube, Medium Time Delay (IEC 60127

5 x 20 mm glass tube, Medium Time Delay (IEC5 x 20 mm glass tube, Medium Time Delay (IEC Time Lag, RoHS compliant.

Time Lag, RoHS compliant.

Time Lag, RoHS compliant.Time Lag, RoHS compliant.

T4A 250V (Time Delay, 4 Amps, 250 Volts)

T4A 250V (Time Delay, 4 Amps, 250 Volts) T4A 250V (Time Delay, 4 Amps, 250 Volts)

Bussmann S506----4444----R or Littelfuse 218

Bussmann S506Bussmann S506

prong). This is required to protect you from injury in the event

prong). This is required to protect you from injury in the event prong). This is required to protect you from injury in the event

occurs.

occurs.occurs.

R or Littelfuse 218 004.P (or 218

R or Littelfuse 218R or Littelfuse 218

power source; appropriately rated for use with this

power source; appropriately rated for use with this power source; appropriately rated for use with this

004.P (or 218 004.HXP)

004.P (or 218004.P (or 218

60127----2, Sheet III) or

6012760127

2, Sheet III) or

2, Sheet III) or 2, Sheet III) or

004.HXP)

004.HXP) 004.HXP)

Appendix C

Appendix CAppendix C

To avoid injuring your back or limbs it is recommended that you do not attempt to lift this

To avoid injuring your back or limbs it is recommended that you do not attempt to lift this To avoid injuring your back or limbs it is recommended that you do not attempt to lift this instrument by yourself. The P

instrument by yourself. The P----1000 Micropipette Puller wei

instrument by yourself. The Pinstrument by yourself. The P and should be moved by TWO (2) people.

and should be moved by TWO (2) people.

and should be moved by TWO (2) people.and should be moved by TWO (2) people.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

1000 Micropipette Puller weighs in excess of 16 kg (over 35 lb)

1000 Micropipette Puller wei1000 Micropipette Puller wei

ghs in excess of 16 kg (over 35 lb)

ghs in excess of 16 kg (over 35 lb) ghs in excess of 16 kg (over 35 lb)

PRE C AUTI O NS

PRE C AUTI O NSPRE C AUTI O NS

Operational Precautions

Operational PrecautionsOperational Precautions

Failure to comply with any of the following precautions may damage this device.

Failure to comply with any of the following precautions may damage this device. Failure to comply with any of the following precautions may damage this device.



Operate the P

Operate the P----1000 using 110

Operate the POperate the P



The P

The P----1000 is designed for operation in a laboratory environment (pollution degree II)

The PThe P and at temperatures between 5°C

and at temperatures between 5°C ---- 40°C.

and at temperatures between 5°C and at temperatures between 5°C



This unit is not designed for operation, nor has it been tested for safety, at altitudes

This unit is not designed for operation, nor has it been tested for safety, at altitudes This unit is not designed for operation, nor has it been tested for safety, at altitudes above 2000

above 2000 meters (6562 feet).

above 2000above 2000



This unit was designed to operate at maximum relative humidity of 80% for temperatures

This unit was designed to operate at maximum relative humidity of 80% for temperatures This unit was designed to operate at maximum relative humidity of 80% for temperatures up to 31°C, decreasing linearly to 50% relative humidity at 40°C.

up to 31°C, decreasing linearly to 50% relative humidity at 40°C.

up to 31°C, decreasing linearly to 50% relative humidity at 40°C.up to 31°C, decreasing linearly to 50% relative humidity at 40°C.



Operate only in a location where there is a free flow of fresh air on all sides. N

Operate only in a location where there is a free flow of fresh air on all sides. NEVER

Operate only in a location where there is a free flow of fresh air on all sides. NOperate only in a location where there is a free flow of fresh air on all sides. N ALLOW THE FREE FLOW OF AIR TO BE RESTRICTED.

ALLOW THE FREE FLOW OF AIR TO BE RESTRICTED.

ALLOW THE FREE FLOW OF AIR TO BE RESTRICTED.ALLOW THE FREE FLOW OF AIR TO BE RESTRICTED.



To avoid burns do not touch the heating filament, the brass clamps holding the filament,

To avoid burns do not touch the heating filament, the brass clamps holding the filament, To avoid burns do not touch the heating filament, the brass clamps holding the filament, or the heated ends of glass pipettes that have been pulled.

or the heated ends of glass pipettes that have been pulled.

or the heated ends of glass pipettes that have been pulled.or the heated ends of glass pipettes that have been pulled.

1000 is designed for operation in a laboratory environment (pollution degree II)

1000 is designed for operation in a laboratory environment (pollution degree II) 1000 is designed for operation in a laboratory environment (pollution degree II)

1000 using 110----220

1000 using 1101000 using 110

meters (6562 feet).

meters (6562 feet). meters (6562 feet).

220V AC., 50

V AC., 50----60Hz

220220

V AC., 50V AC., 50

40°C.

40°C. 40°C.

60Hz----line voltage.

line voltage.

60Hz60Hz

line voltage. line voltage.

EVER

EVER EVER



Only use Sutter Instrument Company replacement heating fi

Only use Sutter Instrument Company replacement heating filaments.

Only use Sutter Instrument Company replacement heating fiOnly use Sutter Instrument Company replacement heating fi

Handling Micropipettes

Handling Micropipettes Handling Micropipettes

Failure to comply with any of the following precautions may result in injury to the users of

Failure to comply with any of the following precautions may result in injury to the users of Failure to comply with any of the following precautions may result in injury to the users of this device as well as those working in the general area near the device.

this device as well as those working in the general area near the device.

this device as well as those working in the general area near the device.this device as well as those working in the general area near the device.



The micropipettes created

The micropipettes created using this instrument are very sharp and relatively fragile.

The micropipettes created The micropipettes created Contact with the pulled micropipette tips, therefore, should be avoided to prevent

Contact with the pulled micropipette tips, therefore, should be avoided to prevent

Contact with the pulled micropipette tips, therefore, should be avoided to prevent Contact with the pulled micropipette tips, therefore, should be avoided to prevent accidentally impaling yourself.

accidentally impaling yourself.

accidentally impaling yourself. accidentally impaling yourself.



Always dispose of micropipettes by placing them into a well

Always dispose of micropipettes by placing them into a well----marked, spill

Always dispose of micropipettes by placing them into a wellAlways dispose of micropipettes by placing them into a well container.

container.

container.container.



Use only with capillary glass (tubing) recommended by Sutter Instrument Company in

Use only with capillary glass (tubing) recommended by Sutter Instrument Company in Use only with capillary glass (tubing) recommended by Sutter Instrument Company in the following section of this manual (

the following section of this manual (1.2.1

the following section of this manual (the following section of this manual (

using this instrument are very sharp and relatively fragile.

using this instrument are very sharp and relatively fragile. using this instrument are very sharp and relatively fragile.

1.2.1).

1.2.11.2.1

).).

laments.

laments.laments.

marked, spill----proof

marked, spillmarked, spill

proof “sharps”

proofproof

“sharps”

“sharps” “sharps”

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

TABLE OF CONTENTS

DISCLAIMER

DISCLAIMER ................................

DISCLAIMERDISCLAIMER

SAFETY WARNINGS AND PRECAUTIONS

SAFETY WARNINGS AND PRECAUTIONS................................

SAFETY WARNINGS AND PRECAUTIONSSAFETY WARNINGS AND PRECAUTIONS

Main Fuse ...............................................................................................................................................iii

Avoiding Electrical Shock or Fire Injury.............................................................................................iii

Back Injury Prevention.........................................................................................................................iii

PRECAUTIONS

PRECAUTIONS ................................

PRECAUTIONSPRECAUTIONS

Operational Precautions .......................................................................................................................iv

Handling Micropipettes.........................................................................................................................iv

1. GENERAL INFORMATION

1. GENERAL INFORMATION ................................

1. GENERAL INFORMATION1. GENERAL INFORMATION

1.1 Introduction.......................................................................................................................................1

1.2 Glass Capillary & Heating Filament Specifications......................................................................1

1.2.1 Glass Capillary............................................................................................................................1

1.2.2 Heating Filament.......................................................................................................................1

1.3 Technical Support.............................................................................................................................2

1.4 System Description – Front Panel..................................................................................................2

1.5 System Description – Mechanical (Puller Anatomy)....................................................................3

1.5.1 Some Basic Information ............................................................................................................3

1.5.2 Air Cooling System.....................................................................................................................3

1.5.3 Heating Assembly ......................................................................................................................4

1.5.4 Upper Pulley Assembly .............................................................................................................5

1.5.5 Cabinet ........................................................................................................................................7

1.5.6 Electronics...................................................................................................................................7

................................................................

...................................................

................................................................

......................................

................................................................

...................iii

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................................................................

...........................................

................................................................

...........1111

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......iii

iii

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iiiiii

iii

iiiiii

..iv

....

iviv

2. INSTALLATION

2. INSTALLATION ................................

2. INSTALLATION2. INSTALLATION

2.1 Unpacking..........................................................................................................................................9

2.2 Setting Up..........................................................................................................................................9

2.2.1 Line Power (Mains)....................................................................................................................9

3. OPERATING INSTRUCTIONS

3. OPERATING INSTRUCTIONS................................

3. OPERATING INSTRUCTIONS3. OPERATING INSTRUCTIONS

3.1 First Time Use ................................................................................................................................11

3.1.1 Quick Start Instructions..........................................................................................................11

3.1.2 Default Configuration..............................................................................................................20

3.2 Programs..........................................................................................................................................22

3.2.1 Program Structure...................................................................................................................22

3.2.2 Program Line Pull Cycle Parameters....................................................................................23

3.2.3 Pull Cycle ..................................................................................................................................24

3.2.4 Start Up.....................................................................................................................................24

3.2.5 Editing a Program....................................................................................................................26

3.2.5.1 Editing Tips........................................................................................................................26

3.2.5.2 Edit User Notes .................................................................................................................26

3.3 Menu screen ....................................................................................................................................28

3.3.1 General......................................................................................................................................28

3.4 System Options ...............................................................................................................................28

3.5 Program Options.............................................................................................................................28

................................................................

..............................................................

................................................................

..............................9999

............................................................

....................................

................................................................

....11

........

1111

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

3.6 Ramp Test........................................................................................................................................29

3.6.1 Heat Value Recommendations ...............................................................................................30

3.7 Pulling Pipettes...............................................................................................................................30

3.7.1 Procedures ................................................................................................................................30

3.7.2 Notes on Program Operation..................................................................................................32

3.8 Parameter Adjustment...................................................................................................................32

3.8.1 General Information................................................................................................................32

3.8.2 Micropipette/Microinjection Needle Fabrication..................................................................33

3.8.2.1 HEAT..................................................................................................................................33

3.8.2.2 PULL Strength..................................................................................................................33

3.8.2.3 VELOCITY (Trip Point)...................................................................................................33

3.8.2.4 TIME Mode (Cooling).......................................................................................................34

3.8.2.5 Delay Mode (Cooling)........................................................................................................34

3.8.2.6 PRESSURE Adjustment ..................................................................................................35

3.8.2.7 Filament Width..................................................................................................................35

3.8.3 Patch Pipette Fabrication .......................................................................................................35

3.8.3.1 HEAT..................................................................................................................................36

3.8.3.2 PULL Strength..................................................................................................................36

3.8.3.3 VELOCITY (Trip Point)...................................................................................................36

3.8.3.4 TIME Mode (Cooling).......................................................................................................36

3.8.3.5 PRESSURE........................................................................................................................36

3.8.4 Thermolock™ Pre-Heat Mode ................................................................................................37

3.8.5 Step-by-Step Patch Pipette Programming............................................................................39

3.9 Technical Tips for Pulling Micropipettes.....................................................................................41

3.9.1 Regulating the Time it takes to pull a Sharp Pipette ..........................................................41

3.9.1.1 HEAT..................................................................................................................................41

3.9.1.2 Pipette Position .................................................................................................................41

3.9.2 Regulating the Length and Tip Size of a Sharp Pipette......................................................41

3.9.2.1 HEAT..................................................................................................................................41

3.9.2.2 Filament Width..................................................................................................................41

3.9.2.3 Air Flow..............................................................................................................................42

3.9.2.4 DELAY Mode of Active Cooling.......................................................................................42

3.10 Fire Polishing................................................................................................................................42

3.11 Heating Filaments........................................................................................................................43

3.11.1 General Information..............................................................................................................43

3.11.2 Trough Filament....................................................................................................................44

3.11.2.1 Positioning .......................................................................................................................44

3.11.2.2 Geometry..........................................................................................................................44

3.11.3 Box Filament ..........................................................................................................................45

3.11.3.1 Positioning .......................................................................................................................45

3.11.3.2 Geometry..........................................................................................................................46

4. MAINTENANCE

4. MAINTENANCE ................................

4. MAINTENANCE4. MAINTENANCE

4.1 Cleaning ...........................................................................................................................................47

4.2 Heating Filament Replacement ....................................................................................................47

4.2.1 Systematic Filament Replacement.........................................................................................47

4.2.2 Air Jet Position.........................................................................................................................47

4.2.3 Positioning the Filament in Relation to the Glass Capillary ..............................................47

4.3 Pulley Adjustment ..........................................................................................................................49

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

................................................................

............................................................

................................................................

............................47

........................................................

4747

vii

4.4 Regeneration of Drierite Granules................................................................................................50

5. TROUBLESHOOTING

5. TROUBLESHOOTING................................

5. TROUBLESHOOTING5. TROUBLESHOOTING

5.1 Controlling Pipette Tip Shapes.....................................................................................................53

5.1.1 Problem: What type of glass should be used? .......................................................................53

5.1.2 Problem: The resistance of pulled pipettes is too low. How can a higher-resistance

pipette be pulled?...............................................................................................................................53

5.1.3 Problem: How can tips be made even smaller?.....................................................................53

5.1.4 Problem: How can the size of a patch-pipette be increased?...............................................54

5.1.5 Problem: Patch-pipette tips vary in size from pull to pull...................................................54

5.1.6 Problem: An injection pipette with a 1mm tip that is 20 to 50mm long needs to be

formed. How can this be done?........................................................................................................54

5.1.7 Problem: The electrodes are bent. How can they be made to pull straight?.....................54

5.1.8 Problem: One electrode is much longer than the other electrode......................................54

5.1.9 Problem: The shape and resistance of the pipette changes from pull to pull....................55

5.2 System Operation/Function Problems .........................................................................................55

5.2.1 Startup Problems.....................................................................................................................56

5.2.2 General Operational Problems...............................................................................................56

5.2.3 On-Screen Emergency Reset Messages.................................................................................56

5.2.4 On-Screen Error Messages (Edit/Pull Screen)......................................................................57

5.3 Diagnostics.......................................................................................................................................59

5.3.1 Air Pressure System ................................................................................................................60

5.3.1.1 Air Pressure Amount (“Pressure dialed in”)..................................................................60

5.3.1.2 Air Pressure Transducer Reading...................................................................................60

5.3.1.3 Air Pump Test (“Run the pump unregulated 1 sec.”)...................................................60

5.3.1.4 Automatic Pressure On/Off..............................................................................................60

5.3.1.5 Air Valve (“Open/close the Air Valve”)...........................................................................61

5.3.1.6 Test for air leaks................................................................................................................61

5.3.1.7 Trace and correct leaks.....................................................................................................61

5.3.2 Heat Diagnostics ......................................................................................................................61

5.3.3 Pull Diagnostics........................................................................................................................62

5.3.4 Velocity Diagnostics.................................................................................................................63

5.4 Touchscreen Calibration................................................................................................................65

5.5 Technical Support...........................................................................................................................66

................................................................

..................................................

................................................................

..................53

....................................

5353

APPENDIX A. LIMITED

APPENDIX A. LIMITED WARRANTY

APPENDIX A. LIMITED APPENDIX A. LIMITED

APPENDIX B. ACCESSOR

APPENDIX B. ACCESSORIES

APPENDIX B. ACCESSORAPPENDIX B. ACCESSOR

APPENDIX C. FUSE REP

APPENDIX C. FUSE REPLACEMENT

APPENDIX C. FUSE REPAPPENDIX C. FUSE REP

APPENDIX D. TECHNICA

APPENDIX D. TECHNICAL SPECIFICATIONS

APPENDIX D. TECHNICAAPPENDIX D. TECHNICA

APPENDIX E. DRIERITE

APPENDIX E. DRIERITE MATERIAL SAFE

APPENDIX E. DRIERITEAPPENDIX E. DRIERITE

INDEX

INDEX................................

INDEXINDEX

................................................................

WARRANTY................................

WARRANTYWARRANTY

IES ................................

................................................................

IESIES

................................................................

LACEMENT................................

LACEMENTLACEMENT

L SPECIFICATIONS................................

L SPECIFICATIONSL SPECIFICATIONS

MATERIAL SAFETY DATA SHEET

MATERIAL SAFE MATERIAL SAFE

................................................................

TY DATA SHEET................................

TY DATA SHEETTY DATA SHEET

................................................................

............................................................

................................................................

...........................................................

................................................................

............................67

........................................................

.........................................

................................................................

...........................71

......................................................

...........................................

................................................................

..................................................

................................................................

.................................................

................................................................

.........69

..................

...........73

......................

..................75

....................................

.................77

..................................

6767

6969

7171

7373

7575

7777

TABLE OF FIGURES

Figure 1-1. P-1000 front panel. .................................................................................................................2

Figure 1-2. P-1000 base plate with components detail........................................................................... 3

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

viii

Figure 1-3. Filament block assembly........................................................................................................4

Figure 1-4. Heat sensor location on filament block assembly. ..............................................................4

Figure 1-5. Upper cable pulley assembly..................................................................................................6

Figure 2-1. P-1000 Cabinet (rear view), detailing power entry module, fuse-holder, fuse, and spare

fuse holder. ..............................................................................................................................10

Figure 2-2. P-1000 Cabinet (end view, left), showing power switch...................................................10

Figure 3-1. Startup screen. ......................................................................................................................11

Figure 3-2. Home screen..........................................................................................................................11

Figure 3-3. Using the knob or keypad to select a program..................................................................12

Figure 3-4. Instruction to run ramp test................................................................................................12

Figure 3-5. Running the ramp test.........................................................................................................13

Figure 3-6. Ramp Test screen. ................................................................................................................13

Figure 3-7. Loading a piece of glass........................................................................................................14

Figure 3-8. Using the PULL button to start the ramp test.................................................................14

Figure 3-9. Selecting a ramp value. ........................................................................................................15

Figure 3-10. Identifying the installed ramp value. ...............................................................................15

Figure 3-11. Using the installed ramp value for Heat..........................................................................16

Figure 3-12. Pressing PULL on front panel to start the ramp test. ...................................................17

Figure 3-13. Cycling through a looped 1-line program.........................................................................17

Figure 3-14. Brief display of Pull Results...............................................................................................18

Figure 3-15. Making a different type of pipette using the Sutter Cookbook. ....................................18

Figure 3-16. Placing puller into Safe Heat Mode..................................................................................19

Figure 3-17. Obtaining Help information..............................................................................................19

Figure 3-18. Examining the contents of a program using the Program Edit screen........................22

Figure 3-19. Home screen........................................................................................................................25

Figure 3-20. Examining the contents of a program using the Program Edit screen........................25

Figure 3-21. Program list screen.............................................................................................................26

Figure 3-22. Edit user notes. ...................................................................................................................27

Figure 3-23. Menu screen. .......................................................................................................................28

Figure 3-24. Ramp test ramp value screen............................................................................................29

Figure 3-25. Pull cycle report screen. .....................................................................................................31

Figure 3-26. Sample program..................................................................................................................31

Figure 3-27. Pull cycle report (multiple-loops)......................................................................................32

Figure 3-28. Side view of fire polishing spacer. .....................................................................................43

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Figure 3-29. Fire-polish spacer in puller bar. ........................................................................................43

Figure 3-30. End view of trough filament and glass.............................................................................44

Figure 3-31. End view of box filament. ..................................................................................................45

Figure 4-1. V-Groove bearings and puller bars. ....................................................................................47

Figure 4-2. Filament alignment. .............................................................................................................48

Figure 4-3. Micropipette shapes..............................................................................................................49

Figure 4-4. Pulley adjustment.................................................................................................................49

Figure 5-1. Starting Diagnostics from the Main Menu screen............................................................59

Figure 5-2. Diagnostics screen.................................................................................................................59

Figure 5-3. Air pressure system diagnostics screen..............................................................................60

Figure 5-4. Heat system diagnostics screen...........................................................................................61

Figure 5-5. Heat system diagnostics error reporting............................................................................62

Figure 5-6. Pull diagnostics screen.........................................................................................................63

Figure 5-7. Velocity diagnostics screen. .................................................................................................64

Figure 5-8. Starting Calibrate TouchScreen from the Main Menu screen. .......................................65

Figure 5-9. TouchScreen Calibration. ....................................................................................................65

Figure 5-10. Power entry module and fuse holder................................................................................71

TABLE OF TABLES

Table 3-1. Standard factory configuration.............................................................................................20

Table 3-2. Trough filament sizes.............................................................................................................44

Table 3-3. Box filament sizes...................................................................................................................46

Table 5-1. Startup Problems Troubleshooting......................................................................................56

Table 5-2. General operational problem troubleshooting. ...................................................................56

Table 5-3. On-screen emergency reset messages. .................................................................................56

Table 5-4. On-screen error message in Edit/Pull Screen when Ramp value is not specified...........57

Table 5-5. On-screen error messages in Edit/Pull screen when PULL is pressed. ...........................58

Table 5-6. Other on-screen error messages in Edit/Pull screen. .........................................................58

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

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P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

1. G E NERA L IN F ORMA TION

GEN E RAL INFO RMAT I ON

1.1.

GEN E RAL INFO RMAT I ONGEN E RAL INFO RMAT I ON

1.1

1.1 Introduction

Introduction

1.11.1

IntroductionIntroduction

The P-1000 can fabricate pipettes for use in intracellular recording, patch clamping, transferring (ICSI, ES Cells), microinjection, aspiration, and microperfusion. Realizing the full potential of this instrument is dependent on a complete understanding of the way it implements the pulling process. To this end, we urge that this manual be read in its entirety. To aid in understanding the function of the instrument, sample programs are already loaded in memory (Programs 0 through 5, as discussed in subsequent material).

The Model P-1000 Flaming/Brown Micropipette Puller combines a proven pulling technology with programmability to produce a very versatile instrument. The pulling mechanism is derived from the Flaming/Brown series of pullers, which have demonstrated the ability to pull a complete range of pipette profiles. Added to this mechanism is the ability to program different pulling sequences; thus, allowing ease of use for pulling a variety of pipette types on one device.

The P-1000 is a ‘velocity sensing’ puller. This patented feature allows the puller to indirectly sense the viscosity of the glass, giving the P-1000 the ability to pull pipettes from all glasses except quartz. Even difficult to pull formulations, such as aluminosilicate glasses, are handled with relative ease.

Throughout this manual reference will be made to the size of the glass tubing used to pull micropipettes. The convention used here for describing the outside diameter (O.D.) and inside diameter (I.D.) is as follows: O.D. x I.D. These dimensions will always be given in millimeters (mm). See our catalog or visit capillaries available for purchase.

1.2

1.2 Glass Capillary & Heating Filament Specifications

Glass Capillary & Heating Filament Specifications

1.21.2

Glass Capillary & Heating Filament SpecificationsGlass Capillary & Heating Filament Specifications

1.2.1

1.2.1 Glass Capillary

Glass Capillary

1.2.11.2.1

Glass CapillaryGlass Capillary

The P-1000 micropipette puller is designed for use with aluminosilicate, borosilicate or other lower melting-point glass tubing or rod ranging from 0.6 to 3.0 mm in diameter. This instrument does not pull quartz glass. Examples of the specific types and sizes of glass that can be used with the P-1000 are listed in the Sutter Instrument Company catalogue that was included with this instrument or can be viewed on Sutter Instrument’s web site at

www.sutter.com

1000.

1.2.2

1.2.2 Heating Filament

Heating Filament

1.2.21.2.2

Heating FilamentHeating Filament

The type and size of glass that you choose may require a Heating Filament other than the one installed in your puller at the factory. Please refer to the Heating Filament section of this manual to determine the appropriate style and size of filament necessary for pulling the specific glass you would like to use. This instrument is designed to accommodate any of the Sutter Instrument Trough-type or Box-type filaments that are shown in the Sutter Instrument catalogue. This selection of replacement filaments can also be viewed on Sutter Instrument’s web site at

. Any glass with comparable technical specifications can be used with the P-

www.sutter.com

to find a wide selection of glass

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

NOTE: USE ONLY SUTTER INSTRUMENT

NOTE: USE ONLY SUTTER INSTRUMENT----SUPPLIED REPLACEMENT HEATING

NOTE: USE ONLY SUTTER INSTRUMENTNOTE: USE ONLY SUTTER INSTRUMENT FILAMENTS IN THIS INSTRUMENT!

FILAMENTS IN THIS INSTRUMENT!

FILAMENTS IN THIS INSTRUMENT!FILAMENTS IN THIS INSTRUMENT!

SUPPLIED REPLACEMENT HEATING

SUPPLIED REPLACEMENT HEATING SUPPLIED REPLACEMENT HEATING

Systematic instructions for replacing the heating filament can be found in the Maintenance section of this manual and in the Help Menu of the P-1000.

1.3

1.3 Technical Support

Technical Support

1.31.3

Technical SupportTechnical Support

Unlimited technical support is provided by Sutter Instrument Company at no charge to our customers. Our technical support staff is available between the hours of 8:00 AM and 5:00 PM (Pacific Standard Time) at (415) 883-0128. You may also Email your queries to

info@sutter.com

1.4

1.4 System Description

System Description –––– Front Panel

1.41.4

System Description System Description

Front Panel

Front Panel Front Panel

Figure 1-1. P-1000 front panel.

Color Touchscreen

Color Touchscreen Provides a user interface with touch sensitive buttons and text

Color TouchscreenColor Touchscreen

descriptions. All touch-sensitive items are blue, including text in blue.

A keypad or keyboard is displayed where needed

Reset

Reset The Reset button is used to perform an emergency reset of the P-

ResetReset

1000’s microprocessor.

Pull Start/Stop

Pull Start/Stop Starts and stops the pull cycle

Pull Start/StopPull Start/Stop

Rotary Dial

Rotary Dial Used as an alternative to the displayed keypad for numeric entry.

Rotary DialRotary Dial

Turn right (clockwise) to increase the currently displayed value, left (counterclockwise) to decrease, and press in to enter the currently displayed value.

Rotating the dial also allows scrolling up or down a list of choices.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

1.5

1.5 System Description

System Description –––– Mechanical (Puller Anatomy)

1.51.5

System Description System Description

1.5.1

1.5.1 Some Basic Information

Some Basic Information

1.5.11.5.1

Some Basic InformationSome Basic Information

Mechanical (Puller Anatomy)

Mechanical (Puller Anatomy) Mechanical (Puller Anatomy)

This section presents a basic mechanical description of the P-1000, with particular emphasis on terminology. Knowing the names of the various parts greatly facilitates communication between the investigators and the manufacturer when discussing adjustments or service problems. In addition, various controls and adjustments on the top of the instrument are located and described. Those adjustments, which are considered part of maintenance procedures, are dealt with in the Maintenance Section of this manual.

1.5.2

1.5.2 Air Cooling System

Air Cooling System

1.5.21.5.2

Air Cooling SystemAir Cooling System

The Model P-1000 supplies a blast of air to cool the filament area after the heating segment of a pull cycle. The components of the air-cooling system are shown below.

Air Compressor Drierite Canister

Air Jet Air Valve Solenoid

Figure 1-2. P-1000 base plate with components detail.

Air Compressor

Air Compressor The air compressor (or pump) creates the air pressure used to cool

Air CompressorAir Compressor

the filament and glass during the pull cycle.

Air Jet

Air Jet Directs the cooling air to the filament. The air jet should be

Air JetAir Jet

positioned 2 to 3 millimeters below the filament. The screw that secures the air jet to the filament block can be loosened allowing the jet to move up and down.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Air Va

Air Valve Solenoid

Air VaAir Va

Drierite Canister

Drierite Canister A canister filled with Drierite used to remove moisture from the air

Drierite CanisterDrierite Canister

lve Solenoid Regulates the flow of cooling air to the filament and glass.

lve Solenoid lve Solenoid

flowing between the pump and the valve solenoid. The desiccated air is used to cool the air flowing to the filament and glass during the pull cycle and to purge heat before and after a pull.

1.5.3

1.5.3 Heating Assembly

Heating Assembly

1.5.31.5.3

Heating AssemblyHeating Assembly

The Heating Assembly

Heating Assembly comprises the Filament, Filament Block Assembly

Heating AssemblyHeating Assembly

Control Chamber

Control Chamber. The Filament Block Assembly and the Humidity Control Chamber are

Control ChamberControl Chamber

Filament, Filament Block Assembly and the Humidity

Filament, Filament Block AssemblyFilament, Filament Block Assembly

Humidity

Humidity Humidity

discussed below. Filaments are discussed in a separate section.

Humidity Control

Humidity Control Humidity Control Chamber

Chamber

ChamberChamber

((((

Figure

Figure 1111----3333

FigureFigure

))))

The chamber encloses the filament block assembly to provide a controlled environment in which to pull the glass. Access holes in the side of the chamber allow the glass to be loaded into position. The chamber must be removed to access the heating filament. To remove, unscrew the thumbscrew on the front plate of the chamber, remove the front plate, and then pull the chamber towards you.

Figure 1-3. Filament block assembly.

Figure 1-4. Heat sensor location on filament block assembly.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Filament Block

Filament Block Filament Block Assembly

Assembly

AssemblyAssembly

((((

Figure

Figure 1111----3333

FigureFigure

Eccentrics and

Eccentrics and Eccentrics and Angle Plate

Angle Plate

Angle PlateAngle Plate

((((

Figure

Figure 1111----3333

FigureFigure

))))

The filament block assembly is made up of several pieces of hard black nylon. Wires supplying current to the filament are attached to threaded ‘posts’. This current is carried to the filament via the upper and lower Brass Jaws and down by loosening the screws (D)

Brass Jaws. Note that these jaws may be moved up

Brass JawsBrass Jaws

(D) that secure them to the front

(D)(D) of the filament block assembly. When changing the filament type from trough to box (or vice versa), the jaws must be moved up or down so that the filament is positioned at the correct level relative to the glass. If the jaws are repositioned and/or the filament type has been changed, make sure that the securing screws of the jaws and filament clamp are again tightened. Failure to tighten these screws can result in, poor current flow for scorching and insufficient heat to melt the glass. Please refer to Systematic Filament Replacement in the Maintenance chapter for additional instructions.

The Angle Plate secures the Filament Block Assembly to the Cover Plate; it contains two eccentric adjustments. The two chromeplated screws AAAA and BBBB are the eccentrics, and A’

A’ and B’

A’A’

B’ are the

B’B’ corresponding locking screws. By turning the eccentrics with a screwdriver the Filament Block Assembly can be moved up and down (A)

(A) or forward and back (B)

(A)(A)

(B) to adjust the position of the

(B)(B) filament. Loosen the locking screw associated with each ‘eccentric screw’ before turning, and tighten after completing the adjustment. Note: Changing the eccentrics should be made only for fine/small adjustments.

Cover Plate

Cover PlateCover Plate

((((

Figure

Figure 1111----3333

FigureFigure

))))

The cover plate conceals the entry of the Pulling Cables into the Base of the instrument. It is attached to the top by two screws, in slots, at points labeled C. Loosening these screws allows the Filament Block/Angle Plate assembly to move forward and back over large distances.

NOTE: The movements of the Cover Plate and the

NOTE: The movements of the Cover Plate and the Jaws constitute the ‘coarse adjustments’

NOTE: The movements of the Cover Plate and the NOTE: The movements of the Cover Plate and the of filament position, while the eccentric screws allow ‘fine adjustments’.

of filament position, while the eccentric screws allow ‘fine adjustments’.

of filament position, while the eccentric screws allow ‘fine adjustments’.of filament position, while the eccentric screws allow ‘fine adjustments’.

1.5.4

1.5.4 Upper Pulley Assembly

Upper Pulley Assembly

1.5.41.5.4

Upper Pulley AssemblyUpper Pulley Assembly

This assembly guides the Pulling Cables 1111----5555)))) to the centrally located (and concealed) Lower Cable Pulley Assembly Upper Cable Pulley Assembly

Upper Cable Pulley Assembly is attached to its panel by two screws, in slots (J’ in

Upper Cable Pulley AssemblyUpper Cable Pulley Assembly

Pulling Cables (T in

Pulling CablesPulling Cables

(T in Figure

(T in (T in

Lower Cable Pulley Assembly. Note that the

Lower Cable Pulley AssemblyLower Cable Pulley Assembly

1111----5555)))), and contains a large eccentric adjustment screw (J in

Jaws constitute the ‘coarse adjustments’

Jaws constitute the ‘coarse adjustments’ Jaws constitute the ‘coarse adjustments’

Figure 1111----5555)))) from the Puller Bars

FigureFigure

(J in Figure

Figure 1111----5555)))). This eccentric screw

(J in (J in

FigureFigure

Puller Bars (G in

Puller BarsPuller Bars

(G in Figure

(G in (G in

(J’ in Figure

(J’ in (J’ in

Figure

FigureFigure

Figure

FigureFigure

is used to adjust cable ‘tension’. Its use is covered in the Maintenance Section, and changes to the settings should not be performed without the supervision of Sutter Instrument Technical Support.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Thi

s cable transmits the pulling force of the solenoid to the Puller

Panels, Left And

Panels, Left And Panels, Left And Righ

Rightttt

RighRigh (K in

(K in Figure

Figure 1111----5555)

(K in (K in

FigureFigure

)

) )

Figure 1-5. Upper cable pulley assembly.

The panels are the angled surfaces that provide mountings for the Puller Bars and their Bearings, the Spring Stops, the Bumpers, and the Upper Cable Pulley Assemblies. Except for minor differences in shape, the left and right Panels are mirror images of each other. Note the three socket-head cap screws that attach each Panel to the base plate top. These screws are used to align the Puller Bars. Their adjustment, if necessary, is covered in the Maintenance Section. Contact Sutter Instrument Technical Support for more instructions on how the panels are aligned.

Bumpers

BumpersBumpers (N in

(N in Figure

Figure 1111----5555))))

(N in (N in

FigureFigure

Spring Stops

Spring StopsSpring Stops (P in

(P in Figure

Figure 1111----5555)

(P in (P in

FigureFigure

Puller Bars

Puller BarsPuller Bars (G in

(G in Figure

Figure 1111----5555))))

(G in (G in

FigureFigure

VVVV---- Bearings

Bearings

Bearings Bearings

(S in

(S in Figure

Figure 1111----5555))))

(S in (S in

FigureFigure

)

) )

The Bumper stops the motion of its associated Puller Bar. Each Bumper also prevents impact forces from breaking pipettes.

The Spring Stops are one-way catches that catch the Puller Bars as they rebound off the Bumpers so as to prevent pipette tip collision.

This assembly consists of the puller bar, threaded post, electrode clamp knob, and cable retaining screw. The cable retaining screw (H)

(H) holds the cable in a shallow groove (I)

(H)(H)

(I) at the end of the puller

(I)(I) bar, and forms the ‘resistance’ against which the cable ends pull. The puller bar is made of mild steel and coated with a controlled thickness of hard chrome. Glass is loaded into the groove near the tip of the puller bar and is held in position by tightening down the clamping knob (R)

(R).

(R)(R)

These bearings are the guides for the Puller Bar motion. They are made of stainless steel and must NEVER

NEVER be oiled (see Maintenance

NEVERNEVER Section). Note that these bearings are mounted on stainless steel bushings, one of which is round with the other two being hexagonal. The hexagonal (eccentric) bushings are used to adjust position and ease of travel of the PULLER BARS (see Maintenance Section). Do not adjust the eccentrics without additional instruction.

Pull Cable

Pull CablePull Cable (T in

(T in Figure

Figure 1111----5555))))

(T in (T in

FigureFigure

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Bars via the Upper (F)

(F) and Lower Pulley Assemblies. It is made of

(F)(F)

flexible metal with a nylon coating. Never pinch or distort the cable. The cable is terminated with crimped-on clamps or ‘swages’ at the back-end of each Puller Bar.

1.5.5

1.5.5 Cabinet

Cabinet

1.5.51.5.5

CabinetCabinet

Baseplate

Baseplate The top thick metal plate on which the mechanical assemblies are

BaseplateBaseplate

mounted.

Base

Base The Base includes the cabinet to which the top Baseplate is

BaseBase

mounted as well as the transformers and the circuit board contained within.

1.5.6

1.5.6 Electronics

Electronics

1.5.61.5.6

ElectronicsElectronics

The P-1000 micropipette puller is controlled by a dsPIC microprocessor. The HEAT power supply is a precision constant-current switching unit. The PULL supply is a constant-current DC power supply. The velocity trip point is set by a D-A converter. The output of the velocity transducer is compared to the output of the velocity D-A to determine when the trip velocity is reached.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

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P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

2. IN STAL LATI O N

INS T ALLA TION

2.2.

INS T ALLA TIONINS T ALLA TION

2.1

2.1 Un

Unpacking

2.12.1

Make certain that you have received all of the following items in the P-1000 shipping box:

  

packing

UnUn

packingpacking

P-1000 micropipette puller*

Power cord

Sample box containing the following types of glass: BF100-50-10 (Microinjection)

BF150-86-10 (Thick-Walled Patch (dissocated or cultured cells))

BF150-110-10 (Thin-Walled Patch (slice or whole tissue))

Four spare heating filaments

Warranty registration

Sutter Instrument Product Catalog



P-1000 & P-97 Pipette Cookbook

The Model P-1000 micropipette puller is shipped to you in a custom box with foam inserts. Please save shipping materials for future use. Should it ever be necessary to ship the puller to another location, the same packaging should be used to prevent damage to the instrument. Additional packing material may be purchased from Sutter Instrument Company.

IMPORTANT: Improper packaging is a form of abuse and, as such, can be responsible for

IMPORTANT: Improper packaging is a form of abuse and, as such, can be responsible for IMPORTANT: Improper packaging is a form of abuse and, as such, can be responsible for voiding the warranty where shipping damage is sustained because of such packing.

voiding the warranty where shipping damage is sustained because of such packing.

voiding the warranty where shipping damage is sustained because of such packing.voiding the warranty where shipping damage is sustained because of such packing.

2.2

2.2 Setting Up

Setting Up

2.22.2

Setting UpSetting Up

2.2.1

2.2.1 Line Power (Mains

Line Power (Mains))))

2.2.12.2.1

Line Power (MainsLine Power (Mains

The P-1000 is designed to operate with a mains power of 110–220 V AC, at 50-60 Hz. The power cord provided with the P-1000 connects the grounded mains power outlet to the Power Entry Module located on the back of the unit (see diagram below). This Module also includes the Line Fuse.

Unless specified otherwise, the P-1000 is shipped equipped with, and pre-programmed for, a 2.5mm box heating filament.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Figure 2-1. P-1000 Cabinet (rear view), detailing power entry module, fuse-holder, fuse, and spare fuse holder.

Make certain that the Power Switch located on the left end of the P-1000 cabinet is OFF (

Figure 2-2. P-1000 Cabinet (end view, left), showing power switch.

OOOO

Plug the power cord provided with the P-1000 into the Line Input socket on the Power Entry Module and then to a power source.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

System Check

3. O P ERAT I NG I NSTR UCTI O

OPER A TIN G IN S TRU C TIO NS

3.3.

OPER A TIN G IN S TRU C TIOOPER A TIN G IN S TRU C TIO

3.1

3.1 First Time Use

First Time Use

3.13.1

First Time UseFirst Time Use

While we realize that most new users of the P-1000 are anxious to start pulling useable pipettes right away, we cannot over-state the importance of taking a few moments to review the manual in order to understand how the puller works. Many a heating filament has been destroyed with first use due to not taking the time to understand the relationship between the programmable heat settings and the filament installed in the puller. If you absolutely must use the puller before reading through the manual, the following instructions are provided to help you get going and keep you from vaporizing your heating filament.

3.1.1

3.1.1 Quick Start Instructions

Quick Start Instructions

3.1.13.1.1

Quick Start InstructionsQuick Start Instructions

1. Remove the rubber bands from the P-1000 puller bar knobs.

2. Plug in the puller to the power AC mains.

3. Turn the puller ON – the power switch is on the left-side panel.

4. The Startup Screen will be displayed for 5 seconds while the puller systems are checked.

NSNS

5. The Home Screen will appear.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Figure 3-1. Startup screen.

Figure 3-2. Home screen.

Use keypad or knob to select Prog. #3

6. Use knob or keypad to select Program # [3]. Press <ENTER> or push IN the knob to enter Program 3.

and then press <ENTER>

Figure 3-3. Using the knob or keypad to select a program.

7. Program 3 will now be displayed.

A “beep” will sound and this message will appear instructing you to run a Ramp Test.

Figure 3-4. Instruction to run ramp test.

8. Press “Ramp” on the touch-screen display to run a Ramp Test.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Figure 3-5. Running the ramp test.

9. Now you will see the Ramp Test Screen.

Press “Ramp” to run a ramp test

Figure 3-6. Ramp Test screen.

The ramp test helps to establish safe heat settings. The ramp value is unknown for the

2.5 x 2.5 box filament (FB255B) filament in your puller and the 1.5 x 0.86 glass you will be using, so you will need to run a Ramp test.

10. Install a piece of 1.5 x 0.86

1.5 x 0.86 capillary glass (BF150

1.5 x 0.86 1.5 x 0.86

BF150----86

BF150BF150

86----10 s

10 sample glass was shipped with

8686

10 s10 s your puller) and ADVANCE THE PULLER BARS ALL THE WAY TOGETHER before tightening the knobs that clamp the glass. Do not over-tighten the knobs.

CAUTION: The program HEAT value should not exceed the listed RAMP TEST value by

CAUTION: The program HEAT value should not exceed the listed RAMP TEST value by CAUTION: The program HEAT value should not exceed the listed RAMP TEST value by more than 10%.

more than 10%.

more than 10%.more than 10%.

The following shows how to load a piece of glass:

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Figure 3-7. Loading a piece of glass.

a. Loosen both clamping knobs (loosening both knobs is recommended so as to avoid

breaking the glass as it is passed through).

b. Place glass in V-groove in puller bar, slide it beyond clamp about 2 cm and tighten

knob.

c. Depress the spring stop on each puller bar to release them from their catch position.

d. Pull both bars towards each other using the finger bars. Hold bars in position using

the thumb and index finger from one hand. The hex head screw should be touching the end of the slot in both puller bars.

e. Loosen both clamping knobs; carefully slide glass through the holes in the side of the

heater chamber and into V-groove of opposite puller bar.

f. Tighten down clamping knobs.

11. Press the “Pull” button on the front panel to start the RAMP TEST.

Press the PULL button on the front panel to start the ramp test

Figure 3-8. Using the PULL button to start the ramp test.

The Ramp Test

Ramp Test will take a few minutes. Please wait: During the ramp test, the filament

Ramp TestRamp Test will gradually heat up until it is hot enough to melt the glass. The glass will not separate, but once the ramp value is reached, there will be a reduction (hour glass-shape) where the glass softened.

12. Once the Ramp Value has been reached, accept the ramp value.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

526

Press here to accept ramp value

Figure 3-9. Selecting a ramp value.

13. The accepted ramp value is now installed in the top right corner of Program 3.

Ramp value installed in Program 3

Figure 3-10. Identifying the installed ramp value.

14. Select Line 1 Heat by touching the screen. Use the keypad or knob to install the ramp value as your heat setting. If you’re ramp test value is 526, install 526 for your Heat. Heat settings within 5 to 10% of the ramp value are the most stable heat settings to use.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

2. Use keypad or knob to install ramp value

1. Press here to highlight

Line 1 Heat.

for Line 1 Heat. Then press ENTER.

3. Heat value is now installed

Figure 3-11. Using the installed ramp value for Heat.

15. Remove the glass that was used for the Ramp test and install a NEW piece of 1.5 x 0.86 glass into the puller bars.

16. With a new piece of glass and the Heat value installed, press the Pull button on the front panel.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Press the PULL button on the front panel to start the Ramp Test

Figure 3-12. Pressing PULL on front panel to start the ramp test.

17. The puller will cycle through this one line program and pull the glass in 4-5 heating cycles (Loops).

Pull in Progress

Figure 3-13. Cycling through a looped 1-line program

18. After 4-5 loops, the glass will separate making two identical pipettes. You will briefly see the pull results in the bottom text box.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Final Pull Results will display for 5 seconds. Press “Menu” then “Pull Results” to review.

Figure 3-14. Brief display of Pull Results.

19. To make a different type of pipette, refer to the program sheet and use one of the six preinstalled programs in your puller (Program # 0-5). If your application or glass and filament combination are not found on the Program Sheet, use the Sutter Cookbook feature found in the menu on the left side of the “Home” screen to search for, and install, the appropriate program settings.

Use Sutter Cookbook feature to search and install programs ideal for your filament, glass, and application.

Figure 3-15. Making a different type of pipette using the Sutter Cookbook.

20. The Safe Heat Mode

Safe Heat Mode helps to prevent burning out the filament. If you install a heat

Safe Heat ModeSafe Heat Mode setting that is 10% over or under the ramp test value, see example below, a Safe Heat Warning

Warning will appear in the text box and disallow that heat setting. Change the heat

WarningWarning

Safe Heat

Safe Heat Safe Heat

setting to avoid damaging or burning out the filament.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Safe Heat

Mode <ON>

Safe Heat Warning!

Figure 3-16. Placing puller into Safe Heat Mode.

21. In the Help Menu, you can access additional features including the Glossary, step-by-step instructions on how to install a filament, and more.

Figure 3-17. Obtaining Help information.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

3.1.2

3.1.2 Default Configuration

Default Configuration

3.1.23.1.2

Default ConfigurationDefault Configuration

Unless special programming or setup was requested at the time of purchase, the puller was setup and shipped with the following standard factory configuration:

Table 3-1. Standard factory configuration.

Heating Filament Installed

Heating Filament Installed 2.5mm Box (Catalog #FB255B)

Heating Filament InstalledHeating Filament Installed

2.5mm Box (Catalog #FB255B)

2.5mm Box (Catalog #FB255B)2.5mm Box (Catalog #FB255B)

Glass used to program puller (sample sent with puller)

Factory installed programs (see enclosed program sheet)

1.0mm O.D. x 0.5mm I.D. borosilicate, without internal filament (catalog #B100-50-10)

0 - Micropipette 0.06 µm tip for high Megohm recording. 1 - Patch type pipette 2 - Microinjection pipette tip < 1 µm 3 - Patch type pipette using 1.5 x 0.86 mm glass 4 - Pronuclear injection pipette using 1.0 x 0.78 mm glass 5 - Patch type pipette using 1.5 x 1.1 mm glass

Additionally, more than 100 programs from the Sutter Pipette Cookbook are stored in the P1000 any one of which is available can be loaded up into any program.

In describing the operation of the puller, the above configuration is assumed. If the configuration of your puller differs, the operating instructions still apply but references to specific program settings may not be accurate. Inappropriate settings will generally only affect your ability to control the geometry of the glass micropipette you are trying to fabricate. However, the heating filament can be destroyed by an excessive va HEAT parameter

HEAT parameter. We recommend you refrain from executing a program until you have read

HEAT parameterHEAT parameter

the heating filament can be destroyed by an excessive value for the

the heating filament can be destroyed by an excessive vathe heating filament can be destroyed by an excessive va

lue for the

lue for the lue for the

this section of the manual, have run the Ramp Test described herein, and are using the Safe Heat Mode.

Program

ProgramProgram (0-99)

A program consists of one or more lines or loops that, when executed, will ‘pull’ the capillary glass inserted in the instrument. A program can be up to 4 lines. Each line can be repeated up to 4 times with a maximum of 10 lines including repeats.

Write Protection

Write ProtectionWrite Protection (Locked/Unlocked)

A program that is Locked cannot be edited or changed.

displayed when the program is locked. A program’s locked status is changed in the Menu screen (displayed when pressing the MENU button).

Safe Heat

Safe HeatSafe Heat (On/Off)

Pressure Setting

Pressure SettingPressure Setting (Range 0-999)

Date/Time

Date/Time Shows the date and time of the last program edit.

Date/TimeDate/Time

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Causes a warning if a Heat value is more than 10% above or below Ramp value.

Shows the programmed value of the air pressure during the active cooling phase of the pull cycle.

No Keypad is

HEAT

HEATHEAT (Range 0-999)

PULL

PULLPULL (Range 0-255)

VELOCITY

VELOCITYVELOCITY (Range 0-255)

HEAT controls the level of electrical current supplied to the filament. The HEAT required to melt a piece of glass is a function of the filament installed and the particular glass size and composition. It is important that the HEAT value be set relative to the Ramp Test value as discussed in the Operation Section. Generally, changes to HEAT will be made in steps of about 5 units since in most cases smaller changes will have little effect.

This parameter controls the force of the hard pull. In general, the higher the PULL, the smaller the pipette’s tip diameter and the longer the taper. Useful changes in PULL strength are 10 units or more to see an effect.

The velocity of the glass carriage system is measured as the glass softens and begins to pull apart under a constant load. The increasing velocity of the initial pull is determined by the viscosity of the glass, which in turn is a function of the glass temperature. The adjustable velocity allows for a selection of a precise glass temperature as the trip point for the hard pull. Useful values for velocity range from 10 to 150 with lower values being used for patch and injection pipettes and higher values for micropipettes. See the Programs section for a discussion of the significance of

Programs section for a discussion of the significance of

Programs section for a discussion of the significance of Programs section for a discussion of the significance of VELOCITY=0.

VELOCITY=0.

VELOCITY=0.VELOCITY=0.

See the

See the See the

TIME

TIMETIME (Range 0-500)

DELAY

DELAYDELAY (Range 0-500)

TIME is one of two available modes of cooling and controls the length of time the cooling air is active. If VEL>0 then one unit of TIME represents 1/2ms. If VEL=0 then one unit of TIME represents 10ms. See the Programs section for a dis

See the Programs section for a discussion of the significance of

See the Programs section for a disSee the Programs section for a dis TIME=0.

TIME=0.

TIME=0.TIME=0.

cussion of the significance of

cussion of the significance of cussion of the significance of

Delay Mode not checked

DELAY is a cooling mode that controls the delay time between when the heat turns off and when the hard pull is activated. The gas is automatically turned on for 300ms. The higher the DELAY value, the cooler the glass will be when the hard pull is executed. Thus, increasing the DELAY results with decreased taper length and increased tip diameter. If VEL>0 then one unit of DELAY represents 1/2ms. If VEL=0 then one unit represents 10ms. See the Programs section for a discussion of the significance of DELAY=0.

section for a discussion of the significance of DELAY=0.

section for a discussion of the significance of DELAY=0.section for a discussion of the significance of DELAY=0.

See the Programs

See the Programs See the Programs

Delay Mode checked

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

3.2

3.2 Programs

Programs

3.23.2

ProgramsPrograms

3.2.1

3.2.1 Program Structure

Program Structure

3.2.13.2.1

Program StructureProgram Structure

The resulting size and shape of a micropipette made using the P-1000 is determined by the parameter values that are programmed by the user. Up to 100 separate programs can be installed and saved. Each program is structured as follows:

Program

Program Consists of one or more Lines, each of which represents a pull cycle.

ProgramProgram

When a program is run, all lines within the program are sequentially executed, beginning with Line 1. As a line is executed, the capillary glass inserted in the instrument is “pulled.” A program can consist of up to 4 lines, and each line can be repeated up to 4 times with a maximum total of 10 pull cycles for the program.

Cycle

Cycle A Cycle

CycleCycle

Cycle consists of six programmable parameters: Line

CycleCycle

Velocity

Velocity, either Tim

VelocityVelocity line of Program code. Each Cycle

Timeeee or Delay,

TimTim

Delay, and Pressure

Delay, Delay,

Cycle can be executed up to four times.

CycleCycle

Pressure. A Cycle

Pressure Pressure

Cycle is equivalent to one

CycleCycle

Line, Heat

Heat, Pull

LineLine

HeatHeat

Pull,

PullPull

A program’s contents can be viewed by displaying the Program Edit screen for the program in question.

Figure 3-18. Examining the contents of a program using the Program Edit screen.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

3.2.2

3.2.2 Program Line Pull Cycle Parameters

Program Line Pull Cycle Parameters

3.2.23.2.2

Program Line Pull Cycle ParametersProgram Line Pull Cycle Parameters

Each of the four programmable parameters in a program line is defined below:

HEAT

HEAT HEAT controls the level of electrical current supplied to the filament.

HEATHEAT

The HEAT required to melt a piece of glass is a function of the filament installed and the particular glass size and composition. It is important that the HEAT value be set relative to the Ramp Test value as discussed in the Operation Section. Generally, changes to HEAT will be made in steps of about 5 units since in most cases smaller changes will have little effect.

PULL

PULLPULL (Range 0-255)

This parameter controls the force of the hard pull. In general, the higher the pull, the smaller the pipette’s tip diameter and the longer its taper. Useful changes in PULL strength are 10 units or more to see an effect.

NOTE: A one-line program containing PULL = 0 and a low VELOCITY setting (15 – 40), looped 2-3 times, can be used for making short patchtype pipettes.

VELOCITY

VELOCITYVELOCITY (Range 0-255)

The VELOCITY of the glass carriage system is measured as the glass softens and begins to pull apart under a constant load. The increasing velocity of the initial pull is determined by the viscosity of the glass, which in turn is a function of the glass temperature. The adjustable velocity allows for the selection of a precise glass viscosity as the trip point for the hard pull. Useful values for velocity range from 10 to 150, with lower values (15-40) being used for patch pipettes and higher values (50-125) for microinjection pipettes. ..

TIME

TIMETIME (Range 0-500)

VELOCITY = 0 Special Condition (Fire Polish Mode):

VELOCITY = 0 Special Condition (Fire Polish Mode): If VEL=0 and

VELOCITY = 0 Special Condition (Fire Polish Mode): VELOCITY = 0 Special Condition (Fire Polish Mode): PULL=0, the HEAT will be on for the duration of the TIME programmed (1 unit equals 10ms). This feature allows you to use the puller to fire polish the resulting patch pipette...

Controls the length of time the cooling air is active. This parameter is one of two available modes of cooling If VEL>0 then one unit of TIME represents 1/2ms. If VEL=0 then one unit of TIME represents 10ms.

TIME = 0 Special Condition:

TIME = 0 Special Condition: The air solenoid is disabled when TIME=0

TIME = 0 Special Condition: TIME = 0 Special Condition: (no active cooling). This allows the pulling of special pipette shapes. Most often used to pull long tube-like shapes such as those used for microperfusion.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

DELAY

DELAYDELAY (Range 0-500)

A cooling mode that controls the delay time between when the HEAT turns off and when the hard PULL is activated. The air is automatically turned on for 300ms. The higher the DELAY value, the cooler the glass will be when the hard PULL is executed. Thus, increasing the DELAY results in decreased taper length and increased tip diameter. If VEL>0 then one unit of DELAY represents 1/2ms. If VEL=0 then one unit represents 10ms.

DELAY = 0 Special Condition:

DELAY = 0 Special Condition: The air solenoid is disabled when DELAY

DELAY = 0 Special Condition: DELAY = 0 Special Condition: = 0 (no active cooling). This allows the pulling of special pipette shapes. Most often used to pull long tube-likes shapes such as those used for aspiration, microperfusion, or holding.

3.2.3

3.2.3 Pull Cycle

Pull Cycle

3.2.33.2.3

Pull CyclePull Cycle

A typical pull cycle in a program line is described below:

1. The heat turns on.

2. The filament heats up, the glass softens, and a weak pull draws the glass out until it reaches the programmed velocity.

3. When the programmed velocity has been reached, the heat turns off, and the air turns on to cool the filament and glass.

4. If TIME is greater than 0 (zero), the hard pull (if any) is executed after a short delay (less than 50ms) and then the air is activated for the specified TIME.

If DELAY is greater than 0 (zero), the air is activated for a short period (less than 400ms) and then the hard pull is activated after the specified DELAY.

3.2.4

3.2.4 Start Up.

Start Up.

3.2.43.2.4

Start Up.Start Up.

After switching on or after pressing RESET, the P-1000 tests the air and heating systems.

The opening screen appears next

Note on Entering Numbers:

Note on Entering Numbers:Note on Entering Numbers:

Numbers can be entered with the on-screen touch Keypad or with the Rotary Dial. Key in all digits for a field (e.g., “012” for a three digit field or key in “12” followed by pressing ENTER. You can also turn the Rotary Dial to increase or decrease a value then press Enter.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Figure 3-19. Home screen.

To open one of the 100 programs, use the touchscreen keypad or the rotary dial to select and enter a program number.

The display will appear as shown below in the following figure.

Figure 3-20. Examining the contents of a program using the Program Edit screen.

On this screen you can:

• Press to open the Menu Screen.

• Press to return to the opening Home screen, where another program can be

opened.

• Press

to erase all parameter values (with the exception of all text in the Notes field), and sets the pressure to a default value of 500. You are asked to confirm the erasure.

• Safe Heat:

Safe Heat:

Safe Heat:Safe Heat:

When checked (

a warning is given if Heat values differ from Ramp by more than 10%. If there is no Ramp value and the Safe Heat is on, the warning is given upon opening the program.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

• Notes:

Notes:

Notes: Notes:

information (e.g., glass and filament for the program, your name, expected results, etc.). To edit, just touch the blue text box and an edit screen with a keyboard will appear.

• Pull pipettes.

Pull pipettes. Once you are satisfied with the settings within the displayed

Pull pipettes. Pull pipettes.

The blue text box at the bottom of the screen can be used to record any

program, you use this program to pull pipettes. To begin the pull process, press the Pull Start/Stop button on the front panel.

An alternate way in which a program can opened for viewing or editing is by pressing the Program List in main menu, and then selecting the program in the list shown in the following screen.

Figure 3-21. Program list screen.

Use the Knob on the front panel to scroll through the list. The currently selected program is shown in green. Press either the OPEN button or the Knob on the front panel to open the program, whereupon the Program Edit screen will be displayed showing the details of the selected program.

3.2.5

3.2.5 Editing a Program

Editing a Program

3.2.53.2.5

Editing a ProgramEditing a Program

On opening a program, the Line 1 Heat value is highlighted with a blue rectangle. You can touch and highlight any parameter for editing. A highlighted parameter can be edited using the Keypad or the Dial. After a number is selected and the ENTER button is pressed, the next field will be highlighted.

3.2.5.1 Editing Tips



All changes to a program cause the “Last Edited” time to update (top of the screen).



A warning is given if a Keypad entry exceeds the maximum for that parameter.



If Safe Heat is checked ( ), a warning is given if a Heat value differs from Ramp by more than 10%.

3.2.5.2 Edit User Notes By touching the blue text box at the bottom of your program you can bring up the “Edit User

Notes” Screen.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Figure 3-22. Edit user notes.

Here you can edit the notes, press [Undo]

[Undo] to start over, or [Clear]

[Undo][Undo]

[Clear] all text and write your own

[Clear][Clear] notes. Pre-installed factory programs (0-5) and any programs imported from the Cookbook feature will have pre-written text notes detailing the filament, glass, pipette type and appropriate heat to use with that program. Take caution before clearing all text, as you might want to retain some information already describing this program. If the notes no longer apply to your program, clear all text and write your own notes.

The “Notes” section is a good place to record whose program it belongs to, the filament type (box or trough), the glass size (OD/ID), and the application for which the program was designed. You might also want to record the heat setting you are using in relation to the Ramp Value. For example: Heat = Ramp +10.

The Rotary Dial allows cursor movement through the text. Cursor position is indicated by the character in which the cursor is positioned turning green.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

3.3

3.3 Menu screen

Menu screen

3.33.3

Menu screenMenu screen

3.3.1

3.3.1 General

General

3.3.13.3.1

GeneralGeneral

Figure 3-23. Menu screen.1

3.4

3.4 System Options

System Options

3.43.4

System OptionsSystem Options

•

Sutter Cookbook:

Sutter Cookbook: Refer to

Sutter Cookbook: Sutter Cookbook:

appropriate program for your application.

•

Set Time and Date:

Set Time and Date: Set Time and Date:

program was installed or last edited.

•

Diagnostics:

Diagnostics: E

Diagnostics: Diagnostics:

find any malfunction.

•

Calibrate Touchscreen:

Calibrate Touchscreen: Calibrate Touchscreen:

calibration is too far off for the touchscreen to work, press RESET and calibrate at the Home screen

• Home Screen:

Home Screen: O

Home Screen: Home Screen:

• Help:

Help: O

Help: Help:

3.5

3.5 Program Options

Program Options

3.53.5

Program OptionsProgram Options

pens the Glossary and Help files

ach of the P-1000 hardware components can be individually operated to

This is the system clock used to record the time of when the

pens the Home screen.

the Cookbook section for information on how to import an

Recalibration may be needed due to drift over time. If

• Ramp Test:

Ramp Test: O

Ramp Test: Ramp Test:

• View Pull Results:

View Pull Results: O

View Pull Results: View Pull Results:

NOTE: The program option “Preheat Jaws 70˚C”in the Menu screen is associated with the Thermolock™ feature, and exists only in P-1000 systems that shipped on and after November 29 of 2010.Also, in the same systems, the Menu screen has both columnar titles ending in “Options”, replacing the previous “Operations”.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

pens the Ramp Test screen

pens a screen that shows details of the last two pulls.

• Lock Program:

Lock Program:

Lock Program: Lock Program:

• Air Be

Air Before Pull:

Air BeAir Be

fore Pull: This function defines the time taken to flush the air subsystem

fore Pull: fore Pull:

with dry air before the pull begins

• Air After Pull:

Air After Pull: Same as above, but after the pull is finished

Air After Pull: Air After Pull:

also 5 seconds.

3.6

3.6 Ramp Tes

Ramp Testttt

3.63.6

Ramp TesRamp Tes

The HEAT value required to melt your glass is based on the characteristics of the heating filament that is installed and the OD/ID of glass you are using. The RAMP TEST allows you to systematically establish or adjust program HEAT values as a function of the filament/glass combination. This test should be run when using the puller for the first time, before writing or editing a program, whenever you change glass or whenever you change the heating filament. A HEAT setting equal to the RAMP value is the best first setting with which to start.

The ramp value is associated and stored with the current program.

You can access the Ramp Test screen from the Menu screen or by touching the Ramp parameter in the Program Edit screen, whereupon the following screen is displayed.

If checked (

), the current program is locked.

. The default setting is 5 seconds.

. The default setting is

Figure 3-24. Ramp test ramp value screen.

If you are using a filament / glass combination for which you have already run a Ramp Test, you can simply type or dial it in. Otherwise, follow the instructions on screen.

1. When the above screen is displayed, press PULL to run the ramp test.

2. The puller increments the HEAT, and the filament, after 1-2 minutes, begins to get hot

and glows orange in color.

3. The puller bars begin to move apart once the HEAT is hot enough to soften the glass.

4. As the glass begins to soften, a certain velocity of the puller bars moving apart is

detected, at which point the heat turns off.

5. Once there is a small melt in the glass, the new ramp value will be displayed.

6. You can accept the ramp value (in which case the value gets installed into your program),

repeat the ramp test, or ignore it and go back.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

7. Accept the ramp value and use a heat-setting equal to, or within five (5) units of, the

ramp test value. This heat setting will be a stable and safe heat to use—one that will not create instability or damage the filament.

3.6.1

3.6.1 Heat Value Recommendations

Heat Value Recommendations

3.6.13.6.1

Heat Value RecommendationsHeat Value Recommendations

For Trough filaments: Recommended starting value: Ramp + 5 units

Maximum program HEAT value(s) = Ramp value + 10%

For Box filaments: Recommended starting value: Ramp value = HEAT setting.

Maximum Program HEAT value(s) = Ramp value + 10%

3.7

3.7 Pulling Pipettes

Pulling Pipettes

3.73.7

Pulling PipettesPulling Pipettes

3.7.1

3.7.1 Procedures

Procedures

3.7.13.7.1

ProceduresProcedures

Prior to pulling a pipette for the first time, it is important to establish what HEAT value is appropriate to melt your glass. Before executing a program for the first time, run the RAMP TEST

TEST as previously described, and accept the Ramp value into the program. The HEAT

TESTTEST

run the RAMP

run the RAMP run the RAMP

settings in the factory-installed programs are typically set at Ramp value. If your Ramp test value differs from the factory Ramp value, adjust the HEAT in your programs to your Ramp value.

Once you have adjusted the HEAT value relative to the Ramp value, pulling a pipette is very straightforward. Try executing the factory installed programs with the sample glass to become acquainted with the pulling process.

1. Load the glass into the puller as described previously in the FIRST TIME USE chapter.

2. Press and then , to open Program 0.

3. Inspect the parameter values displayed for Program 0. Program 0 should display the

factory-installed values listed on the enclosed program sheet. Adjust the HEAT setting to your Ramp value.

4. Press the < PULL > button. The heating filament will turn on and the glass should

separate within 10 seconds. The display will then report the number of heating cycles and the total time that the heat was turned on.

5. Loosen the clamping knobs and remove the pipettes from the puller bars.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Figure 3-25. Pull cycle report screen.

Unless otherwise stated on your program sheet, Program 0 is factory pre-programmed to pull a micropipette (tip diameter less than 0.1mm) from 1.0 x 0.5 mm borosilicate glass. It will pull the pipette in one heating cycle. The time reported is very useful for developing programs and will be discussed in the Parameter Adjustment section of this manual.

A feature of the P-1000 is its capability to loop through a program. This is demonstrated using Program 1, which is a factory pre-installed one-line program “loop” (repeat and pull in stages) to create a patch-type pipette with a short taper and 1-3µm tip. Press RESET to exit

Program 0, and then , to open Program 1. The display for Program 1 should read similar to the following:

Figure 3-26. Sample program.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Load glass into the puller and press <PULL>

<PULL>. The heating filament should loop on and off

<PULL><PULL> repeatedly. Once the glass has separated, the pull is complete, and the display should read similar to the following:

Figure 3-27. Pull cycle report (multiple-loops).

After the heat turns on in line 01, the glass heats up and draws apart until it reaches a VELOCITY of 30, at which point the heat turns off and the cooling air turns on. The puller is “aware” of the fact that the glass has not separated yet, and will go back to line 1 of the program and try again; in effect, it begins ‘looping’. It will continue to do so until the glass separates. This looping capability is particularly useful for fabricating patch pipettes, which require multiple heating cycles to form the characteristic stubby geometry.

Note:

Note: Note:

Heat

Heat----on Time for each line of program executed is recorded.

HeatHeat

3.7.2

3.7.2 Notes on Program Operation

3.7.23.7.2

The results for the last two pipettes pulled are available from the Menu screen. The

The results for the last two pipettes pulled are available from the Menu screen. The The results for the last two pipettes pulled are available from the Menu screen. The

on Time for each line of program executed is recorded.

on Time for each line of program executed is recorded.on Time for each line of program executed is recorded.

Notes on Program Operation

Notes on Program OperationNotes on Program Operation

There is always the possibility that the puller will be given a set of values that ‘stall’ its operation. For example, the HEAT value has not been set high enough to melt the glass, thus the glass cannot be pulled and no VELOCITY can be achieved. If it appears that a situation of this type has arisen, press the STOP key. This action aborts program execution and allows editing to take place. The safe-heat mode (turned on) should prevent this event from occurring.

All programs entered into memory (maximum of 100) remain there even after the power is turned off or the RESET switch is pressed. Nevertheless, it is strongly suggested that you keep a written record of your programs in case of unexpected difficulties.

3.8

3.8 Parameter Adjustment

Parameter Adjustment

3.83.8

Parameter AdjustmentParameter Adjustment

3.8.1

3.8.1 General Information

General Information

3.8.13.8.1

General InformationGeneral Information

Micropipette and microinjection needle programs are sufficiently different from patch pipette programs that the following information on parameter adjustments has been divided into three sections: Micropipette/microinjection needle fabricatio Technical Tips

Technical Tips. Even if your research only requires one type of pipette, we recommend that

Technical TipsTechnical Tips

Micropipette/microinjection needle fabricationnnn, Patch pipette fabrication

Micropipette/microinjection needle fabricatioMicropipette/microinjection needle fabricatio

Patch pipette fabrication and

Patch pipette fabricationPatch pipette fabrication

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

you read all three sections for full appreciation of the capabilities of the puller. Please note that the programs referred to in the following text are not necessarily meant to pull functional pipettes, but are intended as an exercise to help develop an understanding of the programming process. Unless otherwise stated, parameter adjustments assume that the puller is in the TIME

3.8.2

3.8.2 Micropipette/Microinjection Needle Fabrication

Micropipette/Microinjection Needle Fabrication

3.8.23.8.2

Micropipette/Microinjection Needle FabricationMicropipette/Microinjection Needle Fabrication

TIME mode of active cooling (see Software Control Functions).

TIMETIME

Consider the following programs using a 3mm- filament:

Micropipette Program using 1.0mm x 0.5mm borosilicate tubing

HEAT

HEAT PULL

HEATHEAT

Ramp 150 100 150 500

PULL VELOCITY

PULLPULL

VELOCITY TIME

VELOCITYVELOCITY

TIME PRESSURE

TIMETIME

PRESSURE

PRESSUREPRESSURE

Microinjection Needle Program using 1.0mm x 0.75mm borosilicate tubing

HEAT

HEAT PULL

HEATHEAT

Ramp 60 80 200 300

PULL VELOCITY

PULLPULL

VELOCITY TIME

VELOCITYVELOCITY

TIME PRESSURE

TIMETIME

PRESSURE

PRESSUREPRESSURE

3.8.2.1 HEAT The HEAT setting will affect the length and tip size of the pipette. In general, higher HEAT

settings tend to give longer and finer tips. For trough filaments, the recommended starting HEAT value is the ramp test value plus 10%. For box filaments, the recommended starting HEAT value is the ramp test value. The program listed above will typically have heat on for 5 to 8 seconds after the < PULL >

< PULL > button is pressed. If the time is longer than eight seconds,

< PULL >< PULL > and you are trying to pull a fine micropipette, increase the HEAT in 5 unit increments until the pull takes place in less than eight seconds. If the pull occurs in less than three seconds, decrease the HEAT until the pull takes place in 4 to 8 seconds. For the best micropipette reproducibility with the finest tips, you should select a HEAT value that melts the glass in 5 to 6 seconds. For microinjection needles you should select a HEAT value that melts the glass in about 7 seconds or longer.

3.8.2.2 PULL Strength Low values of PULL strength in the range of 40-75 will give larger tips appropriate for

injection needles, while settings between 120-250 give smaller tips appropriate for micropipettes. The PULL strength can be set to any value desired with no danger of damaging the instrument.

3.8.2.3 VELOCITY (Trip Point) The VELOCITY value determines the point at which the heat is turned off. VELOCITY

reflects the speed at which the two carrier bars are moving during the weak pull. The lower the VELOCITY value, the slower the speed of the bars when the trip point occurs. Although VELOCITY can safely be set to any value from 1-255, all values over a maximal trip point (about 150) will produce equivalent results. As the pull progresses, the speed of the carrier bars, as measured by the velocity transducer, reaches a point where further increases in the VELOCITY trip point will not change the time point at which the heat is turned off.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

VELOCITY is typically set between 80 to 120 for micropipettes or 50-80 for microinjection needles.

In a multiple cycle program, it is possible for the glass to separate before the trip velocity is attained. Thus, the glass is subjected to heating as it separates. Such an occurrence can lead to difficulties in forming tips as well as lack of reproducibility. If you are using a one-line, looping program, try decreasing the VELOCITY a few units at a time. If your program is a multi-line program, decrease the VELOCITY in the next to last line of the program. Decreasing the VELOCITY will increase the amount of glass left in the last cycle of the program, thus allowing the glass to attain the trip velocity before separating.

3.8.2.4 TIME Mode (Cooling) The TIME parameter controls the length of time the cooling air is active (one unit of TIME is

equivalent to 1/2msec). In order to produce effective cooling, the air must be supplied to the filament and glass during the time the tip is being formed. In order to guarantee that cooling occurs in this time frame, the start of the hard pull is begun after a fixed delay of less than 50msec from the termination of the heating cycle and the activation of active cooling. In most applications, the hard pull lasts several tens of milliseconds. Because of this fact, increasing cooling TIME values beyond a certain range (typically between 150 to 200) will have no effect. Values of TIME shorter than this same range will cool the glass less as the tip is being formed and lead to a longer taper. However, once TIME values become too short (values in the range 110 to 130) cooling becomes ineffective. The glass will not form a tip and instead forms a wispy fiber. The very finest tips for a given PULL and HEAT will be formed at an air setting of about 5 units higher than the lowest TIME value that forms a tip. Because of this quite narrow working range of usable TIME values for making micropipettes, it is not recommended to vary cooling, and therefore electrode tip length, by adjusting TIME. Adjusting the cooling air pressure, or switching to the Delay

Delay mode of active cooling are both

DelayDelay

more effective means of controlling tip length (see below).

3.8.2.5 Delay Mode (Cooling) In TIME

TIME mode, especially when using larger and or thicker wall glass, active cooling may not

TIMETIME be sufficient to produce short pipette tapers. This may even be true at increased PRESSURE settings (i.e. >500). In this case, it is recommended that the Delay mode of active cooling be used. The method for changing to the Delay mode are described under “Software Control Functions” and a brief description of the two modes of cooling is given in “Programs.”

After switching to DELAY mode, one then has direct control over the delay between turning the heating filament off and initiating of the hard pull. Because the cooling air is turned on when the filament is turned off, increasing DELAY profoundly increases glass cooling before and during the hard pull.

With a range of control over the degree of pipette cooling one can control the rapidity of pipette taper. Longer DELAY values increase cooling and form a pipette with a more rapid taper (shorter shank) while shorter DELAY values decrease cooling and form a pipette with a more gradual taper (longer shank). Fortunately, cooling induced changes in pipette taper generally occur with little effect on tip size. This is quite valuable as the resistance of pipettes is a strong function of the length of the taper. By making a pipette with a sharper taper, one can often decrease pipette resistance significantly without changing the size of the pipette tip. Furthermore, the ability to control tip length is invaluable for experiments where long

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

tips are necessary for penetration into deep tissues or where short stiff tips are necessary for adequate beveling.

Minimum and maximum useable values of DELAY can be expected; their exact values are dependent on glass thickness and diameter. If DELAY is too short, the glass will not cool sufficiently to form a tip and a long wispy fiber of glass will be formed. This minimum DELAY value is typically 80 (less than 50ms of delay). At some maximal value of DELAY, the glass may be cooled too much to separate during the hard pull. Under these conditions, the puller will typically execute multiple cycles in order to separate the glass and the glass may break at a large tip diameter. Maximum usable values of DELAY will be dependent on glass dimensions but are expected to be near 200 (100ms of delay).

3.8.2.6 PRESSURE Adjustment The pressure setting controls the pressure of the cooling air delivered to the filament. The

higher the pressure, the shorter the pipette taper will be. Because thin walled glass cools more rapidly than thick walled glass, the recommended values are ≤ 300 for thin walled tubing and 500 for thick walled or standard walled tubing. By varying PRESSURE around these values, the user can control pipette tip length over a moderate range.

Note that increasing the pressure value to some higher number results in an immediate change in the pressure. A decrease in the pressure value will result in a pressure change at the time of the next pull. This delay in pressure change occurs because the system must first be purged of the air that is pressurized at the higher level. This will only occur prior to the execution of a program. Each time the < PULL >

< PULL > key is pressed the air solenoid is opened

< PULL >< PULL > and air flows through the air jet until the pressure drops below the set value. The solenoid then closes and the compressor will pump until the correct pressure is attained. This pressurization routine is meant to fully cool the filament and assure that the pressure is precisely set.

3.8.2.7 Filament Width Further control over pipette tip length can be accomplished by varying filament width.

Longer tips can be formed by using wider filaments and conversely shorter tips can be formed by using narrower filaments.

3.8.3

3.8.3 Patch Pipette Fabrication

Patch Pipette Fabrication

3.8.33.8.3

Patch Pipette FabricationPatch Pipette Fabrication

Micropipettes used for the electrophysiological recording technique of “patch clamping” are generally characterized by short, stubby shanks and relatively large diameter tips (> 0.7 mm). Programs that can fabricate a pipette with these characteristics generally differ from programs for micropipettes in three ways:

The trip velocity is lower.

The trip velocity is lower. The trip velocity is lower.

The PULL strength is typically turned off.

The PULL strength is typically turned off. The PULL strength is typically turned off.

More than one heating cycle is used.

More than one heating cycle is used.More than one heating cycle is used.

The P-1000 can be used very effectively for this type of processing. The following general information will familiarize you with the effect of adjusting each of the pull cycle parameters in a typical patch pipette program. Following this general information are step-by-step instructions intended to help you establish a stable program to pull patch type pipettes and should be followed in the order described.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Consider the following sample patch program for 1.5 mm O.D. by 1.1 mm I.D. borosilicate glass using a 2.5mm box filament:

HEAT

HEAT PULL

HEATHEAT

Ramp 0 40 250 500

PULL VELOCITY

PULLPULL

VELOCITY TIME

VELOCITYVELOCITY

TIME PRESSURE

TIMETIME

PRESSURE

PRESSUREPRESSURE

A program sequentially executes each line of code, then loops back to the start, and begins again until the glass separates. A single line program such as this may execute 2 to 4 times before the glass separates.

3.8.3.1 HEAT The actual HEAT value used should be sufficiently high to allow the glass to melt in the first

cycle in 5 to 15 seconds. Using a higher HEAT that melts the glass in less than 5 seconds will cause no problem in the first heating cycle, but may heat the glass so much in subsequent heating cycles, that the air cooling will be less effective.

3.8.3.2 PULL Strength A constant gravitational pull on the carrier bars can be felt when loading the glass. This pull

is usually adequate to form relatively small-tipped pipettes (0.5 mm). Eliminating the hard pull from the program (PULL=0) is recommended for most pipettes. If smaller tips are required, a moderate PULL (25-50) may be used in the last line of a multi-line program (see below).

3.8.3.3 VELOCITY (Trip Point) VELOCITY determines the point at which the heat is turned off. If the value is too high, the

glass will separate after the first or second heat cycle. As the VELOCITY is decreased, the amount of glass drawn-out in a given cycle will also decrease, and more cycles will be required to form a tip. The greater the number of cycles, the larger the tip and the shorter the taper will be. However, too many cycles can lead to variability. In general, it is advisable to keep the number of heating cycles to 5 or less.

3.8.3.4 TIME Mode (Cooling) For patch pipette programs, it is advisable to keep the TIME between 200 and 250 to

maximize the cooling of the glass. Delay mode of active cooling is not used in patch pipette fabrication.

By contrast, to micropipette pulling (see above), the TIME settings in the upper range of available values can have a pronounced effect on glass cooling. This additional cooling is effective due to the lack of a hard pull and the multiple cycle nature of the pulling process for patch pipettes.

3.8.3.5 PRESSURE As with micropipette fabrication, the recommended pressure setting when using thick walled

glass is 500 or greater. For thin walled glass, the recommended setting is 300.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

execution of a program. Each time the < PULL >

< PULL > key is pressed the air solenoid is opened

3.8.4

3.8.4 Thermolock™ Pre

Thermolock™ Pre----Heat Mode

3.8.43.8.4

Thermolock™ PreThermolock™ Pre

Heat Mode

Heat ModeHeat Mode

When pulling pipettes where the pull occurs in multiple stages (i.e., patch pipette programs), the temperature of the jaws that hold the filament can range from 23°C to 85°C. This variation in jaw temperature can sometimes create instability in the program and lead to inconsistency in the tip size, taper length, and cone angle of the resulting pipette. To further regulate the pulling conditions, the new Thermolock™ feature can be used to pre-heat and maintain the jaw temperature at 70°C.

If the “mid-point velocity” for writing a stable patch pipette program (Pipette Cookbook, Pg.

30) has been previously established, the shift and rise in jaw temperature is less likely to introduce variability. However, if a program remains unstable where the first few pipettes pulled are not ideal and later pulls provide better electrodes, the Pre-Heat mode for regulating the jaw temperature can make your program more stable and improve the outcome of resulting pipettes.

To activate the Pre-Heat mode in your program, press <MENU> and then select the box to “Pre-Heat Jaws 70°C”.

When using the Pre-Heat mode, air flush durations before and after the pull are increased to 10 sec.

A caution is displayed indicating that the filament will light up and pre-heat. This yellow message bar is displayed when powering up and entering a Pre-Heat program; when waking from sleep; and when going from a non-Pre-Heat program into a Pre-Heat program.

During Sleep mode, the pre-heating is suspended. Upon waking from Sleep, the yellow message bar is displayed.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

The initial pre-heating of the jaws will take a few minutes, during which the jaw temperature is continuously displayed.

The user can load the glass at any time, but if the glass is loaded before the jaws reach 70°C, the PreHeat mode might not function as intended.

When the jaw temperature reaches 70°C, a green message bar is displayed indicating that it is okay to install the glass and pull a pipette.

When the capillary glass is inserted, even if the Pre-Heat temperature has not been reached, the filament heat is turned off to prevent the glass from melting and/or sticking to the filament.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

If the jaw temperature is above 70°C when <PULL> is pressed, the air flush occurs, but the pull cycle is delayed until the jaws cool down to 70°C.

If the jaw temperature has dropped to 70°C after the air flush, the pull cycle will begin immediately.

If a program is opened with the glass already installed, the user is requested to remove the glass to allow the Pre-Heat to continue.

If the glass is inserted when the display is in the Sleep mode, this message is also displayed upon waking from Sleep.

3.8.5

3.8.5 Step

Step----by

by----Step Patch Pipette Programming

3.8.53.8.5

StepStep

Run a Ramp Test with the glass you intend to use for your particular application. Refer to the manual if you need to review the Ramp Test procedure. When you know the Ramp value (R), use it in the following program.

1. Program one line of code as follows:

2. PRESSURE

PRESSURE should be set to 500 for thick walled glass and 300 for thin walled glass.

PRESSUREPRESSURE

****The VELOCITY

Step Patch Pipette Programming

byby

Step Patch Pipette Programming Step Patch Pipette Programming

Filament Type

Filament Type HHHHEAT

Filament TypeFilament Type

Trough Ramp+15 0 *50 150

Box Ramp 0 *30 250

VELOCITY value will need to be manipulated.

VELOCITYVELOCITY

EAT PULL

EATEAT

PULL VELOCITY

PULLPULL

VELOCITY

VELOCITYVELOCITY

TIME

TIMETIME

3. Insert your glass and execute the above program. The program should “loop” a multiple

number of times (i.e. the same line will be repeatedly executed). The display will report the number of loops at the end of the pull sequence. This “looping” is the key to forming

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

patch pipettes. For thin walled glass, 3 to 4 loops are typically all that is required. For thick walled glass, 3 to 5 loops are typically required.

4. Increase the VELOCITY in 5 unit increments and pull a pipette after each adjustment.

Note the change in the number of loops and note the geometry of the pipette (viewed with microscope). As the VELOCITY increases, the number of loops decreases.

5. Repeat step (3) only this time decrease the VELOCITY. As the VELOCITY decreases, the

number of loops increases.

6. By adjusting the VELOCITY as described, establish the number of loops required to

approximately form a pipette with the characteristics you desire. Set the VELOCITY value in your program to the number that falls midway between the values required to loop one more and one less times than the desired number. For example: let’s say that when you are experimenting with VELOCITY values, you find that when the glass separates after 3 loops, the resulting pipette looks pretty reasonable. Let Y be equal to the VELOCITY value that results with the glass separating after 4 loops. Let Z be equal to the VELOCITY value that results with the glass separating after 2 loops. Set your program VELOCITY, to a value midway between Y and Z. This value will be a very stable VELOCITY value and will provide you with the most reproducible results.

7. The one line program just established may be sufficient for your application. However,

changes made in a one-line program are amplified throughout the cycle, potentially producing gross changes in the pipette. If you need to make fine adjustments to the pipette geometry, then you should use a multi-line program. The multi-line program is based on the one line program just established. It is developed as follows:

8. Write your one-line, looping program out into an equivalent multi-line program with the

number of lines equal to the number of loops. For example, a one line, 4-loop program with the following values can be written:

Line

Line Loops

LineLine

Loops HEAT

LoopsLoops

HEAT PULL

HEATHEAT

PULL VELOCITY

PULLPULL

VELOCITY

VELOCITYVELOCITY

TIME

TIMETIME

1 4 285 0 45 150

The above 1-line, 4-loop program could be written into an equivalent 4-line program, as follows:

Line

Line Loops

LineLine

Loops HEAT

LoopsLoops

HEAT PULL

HEATHEAT

PULL VELOCITY

PULLPULL

VELOCITY

VELOCITYVELOCITY

TIME

TIMETIME

1 1 285 0 45 150

2 1 285 0 45 150

3 1 285 0 45 150

4 1 285 0 45 150

9. Now, you can make adjustments to the last or next to last line to fine-tune the program

and the resulting pipette.

10. Recommended changes to fine tune the multi-line program:

For larger diameter tips Decrease HEAT in last line

last line.

last linelast line

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

For smaller diameter tips Increase or decrease VELOCITY in next to last

units, or increase/decrease VELOCITY in la units, or add a small amount of PULL (10 to 20) to last line

3.9

3.9 Technical Tips for Pulling Micropipettes

Technical Tips for Pulling Micropipettes

3.93.9

Technical Tips for Pulling MicropipettesTechnical Tips for Pulling Micropipettes

3.9.1

3.9.1 Regulating the Time it takes to pull a Sharp Pipette

Regulating the Time it takes to pull a Sharp Pipette

3.9.13.9.1

Regulating the Time it takes to pull a Sharp PipetteRegulating the Time it takes to pull a Sharp Pipette

next to last line by 5 to 10

next to lastnext to last

last line

st line by 5 to 10

lala

st linest line

last line.

last linelast line

3.9.1.1 HEAT For 1.0mm O.D. tubing, if the pull takes longer than eight seconds and you are trying to pull

a fine micropipette, increase the HEAT. To do this in a methodical fashion, increase the HEAT value in five unit increments, each time monitoring pull time until the pull takes place in less than eight seconds.

If the pull occurs in less than three seconds after you start, decrease the HEAT value in a similar fashion.

For 2mm O.D. tubing, the pull should occur between 15 and 25 seconds after the start. Make corrections as outlined above for smaller tubing.

3.9.1.2 Pipette Position The position of the glass within the filament will also affect the time it takes to pull a pipette.

When using a trough filament, the glass should be about 0.5mm above the bottom of the filament and centered front to back. In the case of a box filament, the glass should be in the center of the filament. Filament positioning is covered in the next section of this manual “Heating Filaments”.

3.9.2

3.9.2 Regulating the Length and Tip Size of a Sharp Pipette

Regulating the Length and Tip Size of a Sharp Pipette

3.9.23.9.2

Regulating the Length and Tip Size of a Sharp PipetteRegulating the Length and Tip Size of a Sharp Pipette

3.9.2.1 HEAT Higher HEAT settings will give longer and finer tips. A HEAT value equal to the Ramp Test

value plus 15 will generally give a very fine tip.

It should be noted that at high HEAT settings (filament white-hot) the filament life is greatly reduced. It is suggested that a setting equal to the ramp value plus 15 is used initially and electrode length is controlled by air pressure adjustments. If this is insufficient, filament width can be varied.

3.9.2.2 Filament Width Filaments narrower than 2mm cannot form as fine a tip as the wider filaments. The tip size

will decrease with increasing filament width until a width of 3mm is reached. Increasing the filament width beyond 3mm will produce longer tips with a more gradual taper (which may penetrate better in some cases). The tip, however, will not be any smaller.

Tips of 1-2µm can be formed using a 1.5mm filament with low filament temperatures and weak PULL strengths.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

3.9.2.3 Air Flow In general, electrodes will not be formed if the air pressure is set too high. It is thus

recommended that the front panel pressure adjustment be set to standardized values of 500 for thick walled glass and 300 for thin walled tubing. However, as outlined above, under Micropipette/microinjection needle fabrication

Micropipette/microinjection needle fabrication, the length of pipette tips can be controlled by

Micropipette/microinjection needle fabricationMicropipette/microinjection needle fabrication varying air pressure. Furthermore, when making patch pipettes, if increasing TIME to its maximal setting of 255 does not provide enough cooling to produce tips with a short enough taper, then increasing pressure above the standard values may be warranted.

3.9.2.4 DELAY Mode of Active Cooling As discussed previously in this manual, switching from the TIME to the DELAY mode of

active cooling may provide more precise and a wider range of control over the length (or taper) of a sharp pipette tip. The delay mode is often employed when using thick walled glass or for programs designed for the fabrication of pronuclear injection needles.

3.10

3.10 Fire Polishing

Fire Polishing

3.103.10

Fire Polishing Fire Polishing

The P-1000 micropipette puller allows you to perform pseudo fire polishing of pipette tips but does not have a provision for visualizing the pipette tip during the heating process. The extent of the heating required to attain the desired degree of polishing must be empirically established.

What distinguishes a program for polishing from other programs used to pull pipettes is the use of a Velocity value of 0. To program the instrument for the fire-polishing mode, try entering a program as follows:

HEAT

HEAT PULL

HEATHEAT

Ramp value

PULL VELOCITY

PULLPULL

0 0 250

VELOCITY

VELOCITYVELOCITY

TIME

TIMETIME

Pressure does not matter because the air supply is never activated in this mode.

When executed, this program will behave as follows: the Heat will turn on for the duration set by Time. Each Time unit is equivalent to 10 msec. Therefore, in the above program, the Heat will turn on for 2.5 seconds.

The procedure for polishing is as follows:

1. Pull a pair of pipettes with the desired pulling program. After the pipettes have been

pulled, keep them clamped in the puller bars.

2. Reset the puller and select your polishing program (as above).

3. Manually push the puller bar (with the installed pipette) back towards the filament, or

use the Fire Polish Spacer block described next. Try positioning the tip of the pipette just inside the edge of the filament.

4. Press Pull and the filament will heat up for 2.5 seconds, exerting a polish on the end of

the pipette.

How much polishing occurs will be a function of the Heat value and the duration of that Heat as determined by the Time value. You may need to execute the program multiple times to achieve an appropriate polish. You will need to experimentally establish how much Heat and how much Time is necessary for the degree of polishing you require.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

The most difficult part of this procedure is manually positioning each pipette back into the filament at the same relative position each time. The Fire Polishing Spacer allows you to consistently reposition the pipette within the filament. The T-shaped aluminum Spacer has an adjustable setscrew protruding from it.

Figure 3-28. Side view of fire polishing spacer.

The Spacer/screw combination fits into the slot in the puller bar and fixes the puller bar position. You adjust the screw position to set the position of the puller bar (and thus the pipette tip). The more extended the screw is, the closer the pipette tip will be to the heating filament.

When you have finished polishing the pipette, remove the Spacer from the puller bar.

Figure 3-29. Fire-polish spacer in puller bar.

3.11

3.11 Heating Filaments

Heating Filaments

3.113.11

Heating FilamentsHeating Filaments

3.11.1

3.11.1 General Informat

3.11.13.11.1

General Information

General InformatGeneral Informat

ion

ionion

The pipette programs that you ultimately develop will largely depend on the type of heating filament installed in the puller and the glass that you use. Depending on the research application, there may be filament-glass combinations that are more optimal than the one in the current puller setup. After reading through the following material, if you have questions about which filament to use for your application, contact our technical support staff.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

3.11.2

3.11.2 Trough Filament

3.11.23.11.2

Trough Filament

Trough FilamentTrough Filament

Figure 3-30. End view of trough filament and glass.

The trough filament is a general-purpose filament. It is recommended for standard or thin wall glasses used for patch pipette fabrication, sharp electrodes with short tapers, and some types of microinjection needles. The geometry of the trough allows the filament to cool rapidly during the cooling phase of the pulling process.

3.11.2.1 Positioning When using the trough filaments, the glass tubing should be positioned just above the bottom

of the filament (approx. 0.5mm), and centered between the two sides (Figure

Figure 3333----30

FigureFigure

30). This

3030 position can be adjusted by using the two eccentric chrome screws located on the aluminum angle piece that holds the filament assembly (A and B in Figure locking screws (A’

A’ and B’

A’A’

B’ in Figure

B’B’

Figure 1111----3333) and then the filament can be moved in relation to the

FigureFigure

glass tubing by turning the appropriate eccentric chrome screw (AAAA or BBBB in Figure

Figure 1111----3333). First loosen the two

FigureFigure

Figure 1111----3333). See

FigureFigure the Filament Replacement section of the Maintenance Chapter for a full description of this adjustment.

3.11.2.2 Geometry The geometry of the trough filament is an important factor for proper heat application to the

glass. Replacement trough filaments should have a profile similar to that illustrated in Figure

Figure 3333----30

FigureFigure

30, where the distance between the top corners (distance A) is approximately 2/3 the

3030 length of the bottom of the filament. This geometry will provide improved heat distribution to the top of the glass tubing. When replacing a filament, check the new filament geometry. If it differs appreciably from the ideal, you can easily modify it by grasping the bottom corners with non-serrated forceps and gently pushing on the horizontal ‘wings’.

The trough filament you select depends upon the length of the taper that you want. Wider filaments for special purposes are available upon request.

Table 3-2. Trough filament sizes.

Filament

Filament Description

FilamentFilament

FT316B 1.5mm wide trough

FT320B 2mm wide trough

FT330B 3mm wide trough

FT345B 4.5mm wide trough

Description

DescriptionDescription

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

3.11.3

3.11.3 Box Filament

3.11.33.11.3

Box Filament

Box FilamentBox Filament

Figure 3-31. End view of box filament.

Another type of filament that can be used is the box type heater filament. The box filament heats the glass in a more symmetrical fashion than trough filaments, so that the pipettes produced tend to be more straight and concentric than those pulled with a trough filament. It delivers more heat to the glass resulting in faster heating without the necessity of increasing the temperature of the filament.

Note: The Ramp Test value with a box filament will be higher than that with the trough

Note: The Ramp Test value with a box filament will be higher than that with the troughNote: The Ramp Test value with a box filament will be higher than that with the trough filament, thus program HEAT values will be correspondingly higher in order to reach similar

filament, thus program HEAT values will be correspondingly higher in order to reach similar

filament, thus program HEAT values will be correspondingly higher in order to reach similar filament, thus program HEAT values will be correspondingly higher in order to reach similar operating temperatures.

operating temperatures.

operating temperatures. operating temperatures.

Box filaments are recommended for the following micropipettes:

Microelectrodes used for slice preparations where long, parallel walls woul

Microelectrodes used for slice preparations where long, parallel walls would aid

Microelectrodes used for slice preparations where long, parallel walls woulMicroelectrodes used for slice preparations where long, parallel walls woul penetration

penetration

penetrationpenetration

Microinjection needles for transgenic research

Microinjection needles for transgenic researchMicroinjection needles for transgenic research

Microdissection tools

Microdissection toolsMicrodissection tools

Thick or multi

Thick or multi----barreled glass

Thick or multiThick or multi

Aluminosilicate glass

Aluminosilicate glassAluminosilicate glass

barreled glass

barreled glass barreled glass

d aid

d aid d aid

The box filament has two primary limitations.

First, it requires more current to heat to a given temperature than the same size trough filament. Thus, it is possible to use wider trough filaments without exceeding the maximum current capacity of the puller.

Second, the box configuration reduces the cooling effect of the air jet. For this reason the box filament is sometimes not recommended when very short pipettes are to be formed.

3.11.3.1 Positioning When using a box filament, the glass tubing should be centered vertically and horizontally

(Figure

Figure 3333----31

FigureFigure

31).

3131

This position can be adjusted as described above for the trough filament. See Figure

Figure 3333----30

FigureFigure

30 and

3030 the Filament Replacement section of the Maintenance section for a full description of this adjustment.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

3.11.3.2 Geometry The box size you select should be approximately 1.0 to 1.5mm larger than the outside

diameter of the glass you are using. The width of the filament will depend on the research application. A good general-purpose box filament is the 2.5mm wide, 2.5mm high and 2.5mm deep filament (FB255B

FB255B). Special box filaments made to accommodate larger diameter glass or

FB255BFB255B

special pulling applications are available upon request.

Table 3-3. Box filament sizes.

Filament

Filament Description

FilamentFilament

FT215B 2mm square x 1.5mm wide 1.0mm

FT220B 2mm square x 2.0mm wide 1.0mm

FT230B 2mm square x 3.0mm wide 1.0mm

Description Glass O.D.

DescriptionDescription

Glass O.D.

Glass O.D.Glass O.D.

FT255B 2.5mm square x 2.5mm wide

FT245B 2.5mm square x 4.5mm wide

FT315B 3mm square x 1.5mm wide

FT320B 3mm square x 2.0mm wide

FT330B 3mm square x 3.0mm wide

≤ 1.5mm

≤ 2.0mm or 2-3 barrels

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

4. MA INT E NANC E

MAIN TENA NCE

4.4.

MAIN TENA NCEMAI N TEN A NCE

4.1

4.1 Cleaning

Cleaning

4.14.1

CleaningCleaning

To maintain the P-1000 in optimal condition the vinyl dust cover that is shipped with the P1000 should be used whenever the unit is turned off to protect the puller from dust and spills.

Occasionally clean the exterior and the base plate of the unit by wiping them with a dry cloth to remove dust and fine pieces of glass. Avoid contact with the filament.

Figure 4-1. V-Groove bearings and puller bars.

Occasionally the V-groove bearings (S in in their grooves (G in pull. This should be done using a dry cotton swab.

CAUTION: DO NOT lubricate any components of the P

CAUTION: DO NOT lubricate any components of the P----1000!

CAUTION: DO NOT lubricate any components of the PCAUTION: DO NOT lubricate any components of the P

4.2

4.2 Heating Filament Replacement

Heating Filament Replacement

4.24.2

Heating Filament ReplacementHeating Filament Replacement

4.2.1

4.2.1 Systematic Fi

Systematic Filament Replacement

4.2.14.2.1

Systematic FiSystematic Fi

First, remove the chamber that encloses the heating filament. This is done by removing the black thumbscrew at the lower left corner of the Plexiglas cover plate and then pulling the chamber straight off. The old heating filament can then be easily removed by loosening the two clamp screws (D’ in filament, slip in a new one, and center it over the air jet. Then re-tighten the two clamp screws.

4.2.2

4.2.2 Air Jet Position

Air Jet Position

4.2.24.2.2

Air Jet PositionAir Jet Position

The air jet should be from 2-3mm below the center of the filament. If it not within this specification, then loosen the screw holding the air jet in place and reposition it.

G in Figure

Figure 4444----1111) must be cleaned to maintain reproducibility from pull to

G in G in

FigureFigure

lament Replacement

lament Replacementlament Replacement

D’ in Figure

Figure 4444----2222, only one shown

D’ in D’ in

FigureFigure

S in Figure

Figure 4444----1111) and the edges of the pull bars that slide

S in S in

FigureFigure

1000!

1000!1000!

, only one shown) that hold it in place. Slide out the old

, only one shown, only one shown

4.2.3

4.2.3 Positioning the Filament in Relation to the Glass Capillary

Positioning the Filament in Relation to the Glass Capillary

4.2.34.2.3

Positioning the Filament in Relation to the Glass CapillaryPositioning the Filament in Relation to the Glass Capillary

The correct position of the glass capillary in each of the two filament types is shown above in the Heating Filament section. This positioning is critical in achieving the desired pipette tip size and shape; it will also almost certainly require adjustment after replacing a filament. To make this adjustment:

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

1. Carefully slide the glass to be used along the V-groove in the puller bar, and see where it

is positioned relative to the filament.

2. Locate the aluminum angle plate to the right of the filament assembly and behind the

right carrier bar. Two chrome eccentric screws (AAAA and BBBB in Figure

Figure 4444----2222) in slots are

FigureFigure mounted on this bracket, one located on the vertical face of the bracket and one on the horizontal face. Identify the flathead locking screws to the right of each chrome screw.

Figure 4-2. Filament alignment.

3. Loosen the locking screws (A’ and B’)

3.3.

(A’ and B’)

(A’ and B’) (A’ and B’)

4. Turn the eccentric chrome screw (A) on the vertical face to adjust the vertical position of the filament and the eccentric chrome screw (B)

(B) on the horizontal face to adjust the

(B)(B)

front-to-back position of the filament.

5. Tighten the locking screws (A’ and B’)

(A’ and B’)

(A’ and B’)(A’ and B’)

6. If the vertical excursion available with the vertical cam screw isn’t enough to center the glass, you will need to reposition the upper and lower heater jaw assemblies by first loosening the brass screws holding the jaws to the black nylon (D)

(D). Reposition the jaws

(D)(D)

then retighten the brass screws and re-position the air jet.

Testing the Position:

Testing the Position: After positioning the filament it is important to determine if the

Testing the Position: Testing the Position: filament is centered left-to-right over the air jet.

Run a RAMP TEST with your glass and the new filament. If you are unclear as to how to run the ramp test, please review that section of the Control Functions Chapter of this manual.

Program a one-line program similar to the following:

HEAT

HEAT PULL

HEATHEAT

Ramp 120 100 150 500

PULL VELOCITY

PULLPULL

VELOCITY TIME

VELOCITYVELOCITY

TIME PRESSURE

TIMETIME

PRESSURE

PRESSUREPRESSURE

This program is only being used to test pipette length. Pull a pair of pipettes. Remove the pipettes from the carrier bars and hold them side by side as shown in the figure below. If the shanks of the pipettes vary in length, this is an indication that the filament is not centered left to right relative to the air jet, thus one pipette is “seeing” more cooling than the other. Loosen the filament clamping screws and move the filament very slightly towards the side that produced the shorter pipette. Then tighten up the clamps and try another pull. You may have to go through several iterations before you get it centered properly.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

their shoulders and

lengths.

Line pipettes up at

compare their

Right-side pipette is longer than the one on the left. Shift heating filament to the left a small amount (move filament towards shortest pipette).

Figure 4-3. Micropipette shapes.

4.3

4.3 Pulley Adjustment

Pulley Adjustment

4.34.3

Pulley AdjustmentPulley Adjustment

The position of the two pulleys (F in shown)

shown) to the carriers (G in

shown) shown)

(G in Figure

(G in (G in

(F in Figure

Figure 4444----4444A)

(F in (F in

FigureFigure

Figure 4444----4444A)

FigureFigure

A) is adjustable. The pulley position controls the

A)A)

A) that carry the cables from the solenoid (not

A)A)

(not

(not (not

tension of the cables. A difference between the tensions of the two cables can cause problems with pipette reproducibility and/or a disparity between the taper lengths of the pair of pulled pipettes (as illustrated in Figure

Figure 4444----4444). Taper length inequality is generally caused by the air

FigureFigure jet not being aimed at the center of the filament, so to avoid unnecessary adjustments to the pulleys, be certain that the filament and air jet are correctly positioned before proceeding.

The pulley adjustment is made by moving one or both of the pulleys to equalize the tension on the two cables. There are two sets of stops in the system; the stops in the carrier slots against which the carriers rest (M in being pulled out of its housing (not shown).

(M in Figure

(M in (M in

(not shown). The adjustment of the pulleys must be made so

(not shown). (not shown).

Figure 4444----4444A)

FigureFigure

A), and a stop to prevent the solenoid from

A)A)

that the carriers will still come up against their stops while the solenoid is not against its stop. The two cables should not be under high tension when the carriers are against their stops (the position they would be in just before pulling an electrode).

Figure 4-4. Pulley adjustment.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Holding the puller bars together with one hand, you should be able to press on either cable between the carrier and the pulley and feel about 2mm of deflection ((((Figure

Figure 4444----4444B)

FigureFigure

B) before the

B) B) solenoid hits its stop. If the deflection is more or less, the pulley position should be changed. This is done by loosening the two screws above the pulley (J’ in chrome eccentric screws (J in

(J in Figure

Figure 4444----4444A)

(J in (J in

FigureFigure

A) to move the pulley in small increments until the

A)A) two cables are of equal tension. If the carrier no longer stops against its stop in the slot (M in Figure

Figure 4444----4444A)

FigureFigure

A), but stops against the cable, then the cam must be adjusted back until the

A)A)

(J’ in Figure

Figure 4444----4444A)

(J’ in (J’ in

FigureFigure

A) and turning the

A)A)

(M in

(M in (M in

carrier once more hits its stop. It is important that the carriers come up against their stops without significant tension on the cables. If there is too much tension, the initial pull will depend on how tightly you hold the finger stops when the glass is clamped in the carriers. If this happens, the electrodes will not be consistent from pull to pull.

4.4

4.4 Regeneration of Drierite Granules

Regeneration of Drierite Granules

4.44.4

Regeneration of Drierite GranulesRegeneration of Drierite Granules

The Indicating Drierite found in the canister at the right rear corner of the base plate on the P-1000 is a desiccant made of calcium sulfate (97%) and cobalt chloride (3%). This material is used to remove water vapor from the air-cooling supply system. The drierite granules become pink as they absorb moisture, eventually requiring that they be “regenerated” (dried).

Before proceeding, make sure the puller is off and unplug the power cord. To remove the canister from the -, first remove the plastic puller cover by loosening the three screws that hold it down. Next, slide the input (left) and output (right) air tubes off their white plastic connectors on the canister. Finally, the two black plastic clamps that secure the canister to the baseplate can be released by forcing one half of the connector out of the other half at the point where they meet. The canister can be removed.

Unscrew the aluminum end cap, being careful not to loose the black rubber-sealing ring that forms the airtight seal under the cap. With the cap off, the spring, its associated aluminum keeper and the first filter can be removed exposing the Drierite. The exhausted granules can then be removed from the canister. DO NOT UNDER ANY CIRCUMSTANCE REMOVE THE FILTER ON THE RIGHT

THE FILTER ON THE RIGHT. The Drierite should be spread evenly, one granule deep, on

THE FILTER ON THE RIGHTTHE FILTER ON THE RIGHT

DO NOT UNDER ANY CIRCUMSTANCE REMOVE

DO NOT UNDER ANY CIRCUMSTANCE REMOVE DO NOT UNDER ANY CIRCUMSTANCE REMOVE

a tray and heated for one hour at about 200 degrees Celsius. The granules should then be cooled in a tight container before refilling the plastic canister. Filters should also be pre-dried at 100°C for about 30 minutes before assembly. Drierite is not toxic and can be handled with few precautions. For more detailed safety information, please refer to the enclosed MATERIAL SAFETY DATA SHEET.

The Drierite, filters, and keepers are installed in the canister in the order they were removed. First, install a filter against the keeper that was left in the canister. If this keeper was removed, slide it back in first and make sure that it lays flat against the plastic shoulders in the far end of the canister. With the far keeper and filter in place, pour in the regenerated Drierite. Next, insert the second filter followed by the keeper attached to the spring. Finally, lay the rubber seal in the cover and screw on the cover being careful to keep the seal laid out flat as you tighten. There is no need to over tighten the cover, but it should be possible to hear and feel it seating firmly against the rubber seal.

Reinstall the canister on the puller baseplate with its cover to the left and the air tube connections to the front. The black plastic hold-downs slide inside one another and are pushed tight by hand to firmly hold the canister in place. At this tube (larger tube, left

tube (larger tube, left----hand connector) and stop; do not install the output tube.

tube (larger tube, lefttube (larger tube, left

hand connector) and stop; do not install the output tube. Plug in the

hand connector) and stop; do not install the output tube. hand connector) and stop; do not install the output tube.

At this point, install the air input

At thisAt this

point, install the air input

point, install the air input point, install the air input

puller and turn it on. The air pump will turn on and blow air through the canister. Allow this

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

process to continue for several minutes. This procedure allows the purging of any dust or loose particles of Drierite that may have been produced during the recharging process. It is critical that this dust not be blown into the output tube where it might clog either the air

critical that this dust not be blown into the output tube where it might clog either the air

critical that this dust not be blown into the output tube where it might clog either the air critical that this dust not be blown into the output tube where it might clog either the air solenoid or the a

solenoid or the air jet.

solenoid or the asolenoid or the a

ir jet. After the purging process, you may connect the output tube and

ir jet.ir jet.

It is

It is It is

reinstall the puller cover.

If replacement is necessary, Indicating Drierite is manufactured by W.A. Hammond Drierite Co., Ltd. (Xenia, Ohio, USA) and can be purchased from most scientific supply distributors.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

(This page intentionally blank.)

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

5. TR OUBL E SHO O TING

TROU BLES HOOT I NG

5.5.

TROU BLES HOOT I NGTR OUBL E SHOO TING

5.1

5.1 Controlling Pipette Tip Shapes

Controlling Pipette Tip Shapes

5.15.1

Controlling Pipette Tip ShapesControlling Pipette Tip Shapes

5.1.1

5.1.1 Problem: What type of glass should be used?

Problem: What type of glass should be used?

5.1.15.1.1

Problem: What type of glass should be used?Problem: What type of glass should be used?

The type of glass and the wall ratio I.D. (inside diameter) to O.D. (outside diameter) are two of the most important variables in controlling tip size. For example, using borosilicate glass with an O.D. of 1.0mm and an I.D. of 0.50mm will give tips of 0.06 to 0.07mm as demonstrated in Program 0. Using the same settings, borosilicate glass 1.0mm O.D. and

0.78mm I.D. will form tips of 0.1 to 0.12mm. Aluminosilicate glass with an O.D. of 1.0mm and an I.D. of 0.58mm will form tips of 0.03 to 0.04mm again with the same settings.

In general the thicker the wall in relation to the O.D. of the glass the finer the tip will be, and the thinner the wall the larger the tip will be. Thin wall glass will give the best results in most experiments as it will have the largest pore for a given tip size. This means it will have a lower resistance and will allow for easier injection of solutions. However, in many cases with small cells the thin wall glass will not form tips fine enough to obtain good penetrations. In this case, heavier wall glass must be used.

5.1.2

5.1.2 Problem: The resistance of pulled pipettes is too low. How can a higher

Problem: The resistance of pulled pipettes is too low. How can a higher----resistance

5.1.25.1.2

Problem: The resistance of pulled pipettes is too low. How can a higherProblem: The resistance of pulled pipettes is too low. How can a higher pipette be pulled?

pipette be pulled?

pipette be pulled?pipette be pulled?

The first point to note is that there is very little correlation between tip size and electrode resistance. Most of the resistance of a microelectrode is in the shank of the electrode behind the tip. Electrode tips that are 0.1 mm in diameter can vary in resistance from 20MW to 1000MW depending on the length of the electrode and what is used for the filling solution. If the same solution is used then resistance may give an indication of how well the electrode will penetrate a cell as the electrode with the higher resistance will probably have a longer shank and a smaller cone angle at the tip. This combination will aid in the penetration of cells where the cell is not a surface cell.

5.1.3

5.1.3 Problem: How can tips be made even smaller?

Problem: How can tips be made even smaller?

5.1.35.1.3

Problem: How can tips be made even smaller? Problem: How can tips be made even smaller?

1. The first thing to try in most cases is to increase the HEAT value. This will generally

decrease the tip size but it will also give a longer shank. If the higher resistance is not a problem, this is generally the best solution. Continuing to increase the HEAT, however, is not the final answer as too high a HEAT can lead to larger tips. In general, with 1.0mm O.D.X 0.5mm I.D. borosilicate glass the finest tips will be formed when the glass pulls in 5 to 7 seconds after starting the pull.

2. If the electrode is now too long and results in a resistance too high to pass the necessary

current, then the next step is to increase the pull strength. In general, a pull strength of 125 will give tips of less then 0.1mm. Increasing the pull to 250 will reduce tip size about 5-10%. We recommend a pull of about 150 in most cases.

resistance

resistance resistance

3. The last major variable to adjust is the amount of cooling of the glass during the pull. If

in the case of 1.0mm O.D. X 0.5mm I.D. borosilicate glass the pull takes place in 5-7 seconds, the tip size will not change with a change in the cooling air. The only change will be in the length of the shank. If however the HEAT is such that the pull takes place in more then 8 seconds, decreasing the cooling may somewhat decrease the tip size. Cooling

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

can be most effectively decreased in the P-1000 by decreasing air pressure, however a decreasing TIME may also be useful.

5.1.4

5.1.4 Problem: How can the siz

Problem: How can the size of a patch

5.1.45.1.4

Problem: How can the sizProblem: How can the siz

e of a patch----pipette be increased?

e of a patche of a patch

pipette be increased?

pipette be increased?pipette be increased?

1. The first thing to try is to reduce the HEAT. Try dropping the HEAT 5 units at a time to

see if this will increase the size of the tips.

2. If this does not work, increase the pressure in units of 50. The PULL should generally be

set to 0 when pulling large tipped (1-10 mm) pipettes.

3. See also the Step 10 under “Step

ADJUSTMENT

ADJUSTMENT chapter.

ADJUSTMENT ADJUSTMENT

5.1.5

5.1.5 Problem: Patch

Problem: Patch----pipette tips vary in size from pull to pull.

5.1.55.1.5

Problem: PatchProblem: Patch

pipette tips vary in size from pull to pull.

pipette tips vary in size from pull to pull.pipette tips vary in size from pull to pull.

Step----by

by----step patch programming

StepStep

step patch programming” in the PARAMETER

byby

step patch programmingstep patch programming

PARAMETER

PARAMETER PARAMETER

This can happen when a pipette is formed in two or more loops. If the pipette is formed in three loops in one case and then on the next pull it forms in four loops the tips will not be the same. Adding one unit in the VELOCITY value will in most cases cause the pipette to be formed in three loops or subtracting 1 unit should cause the pipette to form in 4 loops. It is always good technique when a program is developed that produces a desired pipette, to try increasing and decreasing the VELOCITY value to be sure that you are in a stable region. The best procedure in developing a very reliable pipette program is to change the VELOCITY value both up and down until the number of cycles to pull the pipette changes. Then pick a value halfway between for the final VELOCITY value.

5.1.6

5.1.6 Problem: An injection

Problem: An injection pipette with a 1mm tip that is 20 to 50mm long needs to be

5.1.65.1.6

Problem: An injectionProblem: An injection formed. How can this be done?

formed. How can this be done?

formed. How can this be done?formed. How can this be done?

pipette with a 1mm tip that is 20 to 50mm long needs to be

pipette with a 1mm tip that is 20 to 50mm long needs to be pipette with a 1mm tip that is 20 to 50mm long needs to be

Try a program in which the first two lines of the program have a PULL value of 0, a VELOCITY value of 10 to 30, a TIME setting of 200 and use the ramp value for the HEAT (box filament). The third line should have the same HEAT value, a PULL value of 150, a VELOCITY of 30 and the TIME should be between 0 to 50 depending on the tip needed (values may vary depending on glass characteristics).

The idea behind this program is to reduce the size of the glass on the first two cycles and then on the third cycle we give a hard pull with the air turned off. Normally if the air is turned off a long wisp will result, but since we have greatly reduced the size of the glass and with a very hard pull the glass will tend to separate when it is about 1mm in diameter.

5.1.7

5.1.7 Problem: The electrodes are bent. How can they be made to pull straight?

Problem: The electrodes are bent. How can they be made to pull straight?

5.1.75.1.7

Problem: The electrodes are bent. How can they be made to pull straight?Problem: The electrodes are bent. How can they be made to pull straight?

This problem occurs most often when using the trough filament. Going to a box type of filament will produce straighter pipettes. The pipette’s bend has no effect on its tip and should not cause problems unless when penetrating quite deeply into tissue and aiming at a certain site. Then the bend may lead the pipette to the wrong area. The box filament is not a complete improvement on the trough filament as the airflow is much less effective with the box filament, and you give up much of the length control that the cooling air gives with the trough filament.

5.1.8

5.1.8 Problem: One electrode is much longer than the ot

Problem: One electrode is much longer than the other electrode.

5.1.85.1.8

Problem: One electrode is much longer than the otProblem: One electrode is much longer than the ot

her electrode.

her electrode.her electrode.

This is caused by one of two things.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

1. The filament may not be centered over the air jet. If it is not, follow the procedure

“Testing the position

Testing the position” in the Heating filament replacement

Testing the positionTesting the position

Heating filament replacement section of the Maintenance

Heating filament replacementHeating filament replacement

chapter.

2. If the filament is correctly centered, then the tension in the two cables that transmit the

pulling force from the solenoid to the puller bars is not equal. To check the tension and adjust if necessary, follow the procedure “Pulley Adjustment

Pulley Adjustment” in the Maintenance

Pulley AdjustmentPulley Adjustment

chapter.

5.1.9

5.1.9 Proble

Problem: The shape and resistance of the pipette changes from pull to pull.

5.1.95.1.9

ProbleProble

m: The shape and resistance of the pipette changes from pull to pull.

m: The shape and resistance of the pipette changes from pull to pull.m: The shape and resistance of the pipette changes from pull to pull.

1. In most cases, this is due to one or both of the cables to the pipette carriers being set up

too tight. If the cable is adjusted so that the carrier can’t come against the stop in the slot in the center of the pipette carrier, then the initial pull tension will depend on how hard the carriers are squeezed together when the glass clamps are tightened. To adjust, see the pulley adjustment section.

2. A second possible cause of this problem is dirt on the carrier bars or bearings. In this

case, clean the carriers and bearings with a lint free tissue or cloth.

3. If the problem persists, then run the ramp test several times. If possible, use one long

piece of glass and move the glass over after each ramp test. If the ramp values are +/- 4 units or less the problem may be with the glass. If the values are greater than +/- 4 units call Sutter Instruments.

5.2

5.2 System Operation/Function Problems

System Operation/Function Problems

5.25.2

System Operation/Function ProblemsSystem Operation/Function Problems

The P-1000 has many built-in automated test capabilities. When the system starts up, the hardware is tested for normal operation and error messages are generated upon detection of a failure. Additionally, automated testing occurs before each pull. Finally, a diagnostics menu screen is provided, allowing for diagnostic testing on demand.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

5.2.1

5.2.1 Startup Problems

Startup Problems

5.2.15.2.1

Startup ProblemsStartup Problems

Table 5-1. Startup Problems Troubleshooting.

Problem on Start Up

Problem on Start Up Possible Causes and Solutions

Problem on Start UpProblem on Start Up

Possible Causes and Solutions

Possible Causes and SolutionsPossible Causes and Solutions

Display is blank or

Display is blank or Display is blank or locked up, and neither

locked up, and neither

locked up, and neither locked up, and neither PULL nor RESET

PULL nor RESET

PULL nor RESET PULL nor RESET switch is illuminated

switch is illuminated

switch is illuminatedswitch is illuminated

1. Check power cord and wall AC power outlet.

2. If the unit still does not work after verifying it is properly

3. If the fuse is still good, the unit is properly plugged in and it still

Display is blank or

Display is blank or Display is blank or locked up and either or

locked up and either or

locked up and either or locked up and either or both PULL and RESET

both PULL and RESET

both PULL and RESET both PULL and RESET switches are flashing

switches are flashing

switches are flashing switches are flashing on/off

on/off

on/offon/off Display is blank and

Display is blank and

Display is blank and Display is blank and both PULL and RESET

both PULL and RESET

both PULL and RESET both PULL and RESET switches are not

switches are not

switches are not switches are not illuminated or flashing

illuminated or flashing

illuminated or flashing illuminated or flashing on/off

on/off

on/offon/off

5.2.2

5.2.2 General Operational Probl

General Operational Problems

5.2.25.2.2

General Operational ProblGeneral Operational Probl

A short exists in the controller’s circuitry. The system must be returned to Sutter Instrument Company for repair.

Press RESET switch to restart the syste4m.

plugged in, remove the power cord and check the fuse. If the fuse has blown, it should be changed. However, if it blows a second time, contact Sutter Instrument Company Technical Support.

does not work, a failure in components that are not serviceable by the user has likely occurred. Contact Sutter Instrument Company Technical Support.

ems

emsems

Table 5-2. General operational problem troubleshooting.

Problem

Problem Possible Causes and Solutions

ProblemProblem

Displayed program

Displayed program Displayed program values are

values are not correct

values arevalues are

not correct

not correct not correct

1. Make sure that values were not changed by another user.

2. If the values entered are not held when the power is turned off,

5.2.3

5.2.3 On

On----Screen Emergency Reset Messages

5.2.35.2.3

Circuit overheated

Circuit overheatedCircuit overheated Please wait for cooldown to 70 C

Please wait for cooldown to 70 C

Please wait for cooldown to 70 CPlease wait for cooldown to 70 C Heatsink temperature is 72 C

Heatsink temperature is 72 C

Heatsink temperature is 72 CHeatsink temperature is 72 C

Screen Emergency Reset Messages

OnOn

Screen Emergency Reset MessagesScreen Emergency Reset Messages

Table 5-3. On-screen emergency reset messages.

Emergency Reset Messages

Emergency Reset Messages Possible Causes and Solutions

Emergency Reset MessagesEmergency Reset Messages

Possible Causes and Solutions

Possible Causes and SolutionsPossible Causes and Solutions

Always write down the program values and the ramp-test value and keep them in a secure place.

a failure in components that are not serviceable by the user has likely occurred. Contact Sutter Instrument Company Technical Support.

Possible Causes and Solutions

Possible Causes and SolutionsPossible Causes and Solutions

Indicates a serious fault in the system, requiring that it cool down. If the problem recurs after allowing sufficient time to cool down, the problem is likely due to a circuit fault, requiring repair at Sutter Instrument Company.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Emergency Reset Messages

Emergency Reset Messages Possible Causes and Solutions

Emergency Reset MessagesEmergency Reset Messages

Air Pump ran 5 minutes continuously.

Air Pump ran 5 minutes continuously.Air Pump ran 5 minutes continuously. Please correct problem and R

Please correct problem and RESET or Open

Please correct problem and RPlease correct problem and R Diagnostics

Diagnostics

DiagnosticsDiagnostics

The Air Pump failed to reach

The Air Pump failed to reach The Air Pump failed to reach pressure

pressure

pressurepressure

The Air Valve failed to open

The Air Valve failed to openThe Air Valve failed to open

There is a major airleak

There is a major airleakThere is a major airleak

There is a fault in the Heat circuit.

There is a fault in the Heat circuit.There is a fault in the Heat circuit.

ESET or Open

ESET or Open ESET or Open

Possible Causes and Solutions

Possible Causes and SolutionsPossible Causes and Solutions

A massive air leak has occurred. Follow the instructions in the error message.

A Serious air leak has occurred. (No leak test was possible.)

A fault has occurred in the air pump or drive circuit.

Electrical disconnection. A fault has occurred in the air valve or drive circuit.

The leak is probably serious enough to affect your pipettes, and if the leak causes air to flow through the Drierite canister, the Drierite will wear out prematurely. Trace the leak using the Help File assistant.

This fault is due to a circuit problem – probably a failed FET (Field Effect Transistor). Run Diagnostics to determine the problem more precisely.

No filament current detected.

No filament current detected.No filament current detected. The Heater Filament may be missing

The Heater Filament may be missing

The Heater Filament may be missing The Heater Filament may be missing or broken.

or broken.

or broken.or broken. If not there is a Heat circuit fault.

If not there is a Heat circuit fault.

If not there is a Heat circuit fault.If not there is a Heat circuit fault.

The filament either is not installed or is burned out. If a working filament is installed, this fault is due to a circuit problem – probably a failed FET (Field Effect Transistor). Run Diagnostics to determine the problem more precisely.

5.2.4

5.2.4 On

On----Screen Error Messages (Edit/Pull Screen)

5.2.45.2.4 If Safe Heat

Safe Heat is checked, Heat values are compared to Ramp. The following error messages

Safe Heat Safe Heat

can occur; follow the instructions in corresponding error message to correct the problem.



If Jaw PreHeat

Jaw PreHeat is checked, a Ramp value is needed. If a Ramp value greater than zero is not

Jaw PreHeatJaw PreHeat

specified, the following error message appears.

Screen Error Messages (Edit/Pull Screen)

OnOn

Screen Error Messages (Edit/Pull Screen)Screen Error Messages (Edit/Pull Screen)

Ramp = 0

Ramp = 0 ----

Ramp = 0 Ramp = 0 risk)

risk)

risk)risk) Line x Heat > Ramp + 15%. Filament may burn out. Reduce Heat or run Ramp

Line x Heat > Ramp + 15%. Filament may burn out. Reduce Heat or run Ramp

Line x Heat > Ramp + 15%. Filament may burn out. Reduce Heat or run Ramp Line x Heat > Ramp + 15%. Filament may burn out. Reduce Heat or run Ramp Test or uncheck Safe Heat

Test or uncheck Safe Heat

Test or uncheck Safe Heat Test or uncheck Safe Heat Line x Heat < Ramp

Line x Heat < Ramp ---- 15%. Jaws may overheat. Increase Heat or run Ramp Test

Line x Heat < Ramp Line x Heat < Ramp or uncheck Safe Heat

or uncheck Safe Heat

or uncheck Safe Heat or uncheck Safe Heat

unable

unable unable

to Heat Check. Press Ramp or uncheck Safe Heat (at your o

to Heat Check. Press Ramp or uncheck Safe Heat (at your own

to Heat Check. Press Ramp or uncheck Safe Heat (at your oto Heat Check. Press Ramp or uncheck Safe Heat (at your o

(at

your risk)

(at(at

your risk) your risk)

15%. Jaws may overheat. Increase Heat or run Ramp Test

15%. Jaws may overheat. Increase Heat or run Ramp Test 15%. Jaws may overheat. Increase Heat or run Ramp Test

(at

your risk)

(at(at

your risk) your risk)

wn wn

Table 5-4. On-screen error message in Edit/Pull Screen when Ramp value is not specified.

Error Message

Error Message Possible Causes and Solutions

Error MessageError Message

A Ramp value is needed to use PreHeat

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Possible Causes and Solutions

Possible Causes and SolutionsPossible Causes and Solutions

If Jaw PreHeat is checked, a Ramp value greater than zero is needed.

When Pull is pressed, the program is checked for validity, and some hardware checks are performed. An erroneous line found in the current program or a failed hardware check can generate one of the following error messages.

Table 5-5. On-screen error messages in Edit/Pull screen when PULL is pressed.

Error Mess

Error Message

Error MessError Mess

age Possible Causes and Solutions

ageage

Possible Causes and Solutions

Possible Causes and SolutionsPossible Causes and Solutions

Line 1 Heat = 0

Line 1 Heat = 0Line 1 Heat = 0

Lines with Heat = 0’. Please edit

Lines with Heat = 0’. Please edit Lines with Heat = 0’. Please edit these lines.

these lines.

these lines.these lines. Puller bars are separated. Reclamp

Puller bars are separated. Reclamp

Puller bars are separated. Reclamp Puller bars are separated. Reclamp glass. Then press PULL again

glass. Then press PULL again

glass. Then press PULL againglass. Then press PULL again

Heat current error. Is Filament

Heat current error. Is Filament Heat current error. Is Filament present and intact? See

present and intact? See

present and intact? See present and intact? See Menu/Diagnostics if Filament is OK

Menu/Diagnostics if Filament is OK

Menu/Diagnostics if Filament is OKMenu/Diagnostics if Filament is OK Failed to reach pressure

Failed to reach pressure

Failed to reach pressureFailed to reach pressure

Failed to melt glass.

Failed to melt glass.Failed to melt glass.

Other error messages.

Obviously not a valid Program.

These Program Lines are not valid.

The puller Bars must be drawn together to the limit of travel.

If they are correctly positioned, go to Menu/Diagnostics to check for a fault condition.

Filament Heater current is not behaving correctly. To distinguish between a broken/missing Filament and a circuit problem, go to Menu/Diagnostics.

There may be an air leak or other problem. Go to Menu/Diagnostics to determine. Also, see Home/Help Files/ Air system diagnostics to trace leaks.

This message shows if the glass does not soften after 60 seconds of heating.

The Filament may have burned out (go to Menu/Diagnostics) or the Heat value may be too low. Perform a Ramp Test and use Safe Heat mode.

Table 5-6. Other on-screen error messages in Edit/Pull screen.

Error Message

Error Message Possible Causes and Solutions

Error MessageError Message

Maximum 10 Program Lines

Maximum 10 Program Lines Too many lines in your Program.

Maximum 10 Program LinesMaximum 10 Program Lines

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Possible Causes and Solutions

Possible Causes and SolutionsPossible Causes and Solutions

5.3

5.3 Diagnostic

Diagnosticssss

5.35.3

DiagnosticDiagnostic

The P-1000’s built-in diagnostics is started by selecting “Diagnostics” in the main menu screen.

Figure 5-1. Starting Diagnostics from the Main Menu screen.

The Diagnostics screen provides the means by which each P-1000’s hardware subsystem can be operated and tested indipendently.

Figure 5-2. Diagnostics screen.

The Diagnostics screen can be invoked at any time should test and diagnostics be desired on any of the four hardware subsystems listed in the menu. These same diagnostics can be useful in troubleshooting problems while in telephone contact with Sutter Instrument Company Tech Support. The rest of this section covers the use of the Air, Heat, Pull, and Velocity Sensor subsystem diagnostics.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

5.3.1

5.3.1 Air Pressure System

Air Pressure System

5.3.15.3.1

Air Pressure SystemAir Pressure System

The Air Pressure System diagnostics menu shown in the following figure lists several diagnostic tests any one of which can be selected for specific tests.

Figure 5-3. Air pressure system diagnostics screen.

5.3.1.1 Air Pressure Amount (“Pressure dialed in”)

The amount of air pressure is shown in the “Pressure dialed in” field. This field is always highlighted so that differrent pressures can be selected using the front-panel Dial. Once the field contains the desired pressure value for testing, press ENTER to set that value.

5.3.1.2 Air Pressure Transducer Reading

The associated non-editable numeric field contains the pressure value from the transducer after a test has been made.

5.3.1.3 Air Pump Test (“Run the pump unregulated 1 sec.”)

Run this test to verify that the air pump is working. While the test is running, the air pump will be audible, and if the test is successful, the pressure transducer reading will rise.

NOTE: The Air Pump Test can be run repeatedly to cau

NOTE: The Air Pump Test can be run repeatedly to cause an increase in air pressure beyond

NOTE: The Air Pump Test can be run repeatedly to cauNOTE: The Air Pump Test can be run repeatedly to cau its normal working range. No harm will result from doing this. However, the noise level

its normal working range. No harm will result from doing this. However, the noise level

its normal working range. No harm will result from doing this. However, the noise level its normal working range. No harm will result from doing this. However, the noise level coming from the air pump will become excessive.

coming from the air pump will become excessive.

coming from the air pump will become excessive.coming from the air pump will become excessive.

se an increase in air pressure beyond

se an increase in air pressure beyond se an increase in air pressure beyond

5.3.1.4 Automatic Pressure On/Off

The Automatic Pressure test is a test of the air pump and the regulating circuitry. This test also checks for air leakage from the system.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

5.3.1.5 Air Valve (“Open/close the Air Valve”)

This test verifies that the air valve opens correctly causing the displayed Pressure Transducer value to drop.

5.3.1.6 Test for air leaks

The air leak test consists of raising the pressure to 500 and then turning off the pump for 10 seconds while testing for a drop in pressure. During the 10 seconds that the pump is turned off, an error will be reported if the pressure drops more than 6 units.

5.3.1.7 Trace and correct leaks

This sequence of tests facilitates the detection and location of leaks in the air system, and provides guidelines on how to repair the leaks. Follow the instructions on the screen.

Tip:

Tip: A good test for full flow through the air valv

Tip: Tip:

A good test for full flow through the air valve and air jet is as follows:

A good test for full flow through the air valvA good test for full flow through the air valv

e and air jet is as follows:

e and air jet is as follows:e and air jet is as follows:

1. Set Pressure to 500.

2. Turn on both Automatic Pressure and Open the Air Valve.

3. The Pressure transducer reading should be 450

If the pressure is significantly lower than 450, the pump is weak and could

If the pressure is significantly lower than 450, the pump is weak and could If the pressure is significantly lower than 450, the pump is weak and could affect the performan

affect the performance of your programs.

affect the performanaffect the performan

5.3.2

5.3.2 Heat Diagnostics

Heat Diagnostics

5.3.25.3.2

Heat DiagnosticsHeat Diagnostics

Set Pressure to 500.

Set Pressure to 500. Set Pressure to 500.

Turn on both Automatic Pressure and Open the Air Valve.

Turn on both Automatic Pressure and Open the Air Valve. Turn on both Automatic Pressure and Open the Air Valve.

The Pressure transducer reading should be 450 ––––500.

The Pressure transducer reading should be 450 The Pressure transducer reading should be 450

ce of your programs.

ce of your programs.ce of your programs.

500.

500.500.

The diagnostics for the heating system is reached by the pressing Heat in the main diagnostics screen, whereupon the screen shown in the following figure is displayed.

Figure 5-4. Heat system diagnostics screen.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

To start the heat diagnostics, press Enter. The heating system is tested at a value of 200 for 10 seconds. The Heat current is controlled by two field effect transistors (FETs). FET A and FET B currents should both be equal to the Heat value of 200, within a few units. If they are unequal, there is circuit board fault. If they are both lower than the Heat setting, the Heat current is low. This can be due to a circuit board fault, but more often indicates a broken heat filament.

Fault conditions are reported in red after the test, as shown in the following figure.

Figure 5-5. Heat system diagnostics error reporting.

The temperatures of the Puller Jaw and the internal Heatsink are continuously displayed. If Jaw Temperature exceeds the maximum allowed, the Heat will be automatically turned off to avoid damage to the nylon mounting block. If the internal heatsink temperature exceeds its maximum, a RESET will occur.

5.3.3

5.3.3 Pull Diagnostics

Pull Diagnostics

5.3.35.3.3

Pull DiagnosticsPull Diagnostics

The Pull diagnostics screen is reached by selecting “Pull” in the main diagnostics screen. The pull diagnostics screen is shown in the following figure.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Figure 5-6. Pull diagnostics screen.

The Pull value is always highlighted for editing with the Dial. Press ENTER to set.

WARNING: With a Dial setting of 25

WARNING: With a Dial setting of 255 (the highest possible), a violent separation of the

WARNING: With a Dial setting of 25WARNING: With a Dial setting of 25 Puller Bars occurs during a Pull, which can cause injury. Use a rubber band rather than your

Puller Bars occurs during a Pull, which can cause injury. Use a rubber band rather than your

Puller Bars occurs during a Pull, which can cause injury. Use a rubber band rather than your Puller Bars occurs during a Pull, which can cause injury. Use a rubber band rather than your fingers to hold the bars together for any value over 100.

fingers to hold the bars together for any value over 100.

fingers to hold the bars together for any value over 100.fingers to hold the bars together for any value over 100.

CAUTION: Never

CAUTION: Never clamp a piece of glass to test a Pul

CAUTION: NeverCAUTION: Never

clamp a piece of glass to test a Pull. Something may get broken as a result.

clamp a piece of glass to test a Pul clamp a piece of glass to test a Pul

5 (the highest possible), a violent separation of the

5 (the highest possible), a violent separation of the 5 (the highest possible), a violent separation of the

l. Something may get broken as a result.

l. Something may get broken as a result.l. Something may get broken as a result.

Press PULL to run the test. The End of Puller bar travel sensor shows “Tripped” when the bars are apart, not when they are fully together.

5.3.4

5.3.4 Velocity Diagnostics

Velocity Diagnostics

5.3.45.3.4

Velocity DiagnosticsVelocity Diagnostics

The velocity diagnostics screen is reached by pressing “Velocity” in the main diagnostics screen. The Velocity diagnostics screen is shown in the following figure.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

Figure 5-7. Velocity diagnostics screen.

In this screen, you can check the sensitivity of the velocity sensor and the operation of the optical sensor that detects when the glass has separated.

The Velocity value is always highlighted for editing with the Dial. Press ENTER to set.

When the Puller Bars move apart, a signal is generated that is dependent on the velocity of separation.

The Velocity setting is the threshold value at which the Trip occurs, as indicated on the screen.

A difference in sensitivity to movement at higher or lower settings can be noticed.

The Background Velocity serves to calibrate the true zero reading. It is typically 1-2 units. A higher number can indicate an electronic fault.

This calibration is done automatically at each pull.

The “Puller bar end of travel sensor” shows “Tripped” when the bars are apart and “Not Tripped” when they are fully together.

Adjust your Program Heat values to reflect the change in Ramp value.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

5.4

5.4 Touchscreen Calibration

Touchscreen Calibration

5.45.4

Touchscreen CalibrationTouchscreen Calibration

The touchscreen can drift out of alignment with the display over time. If ever the screen is so badly misaligned that the Touchscreen is not usable, recovery is still possible. Press RESET. When the Home Screen displays press PULL and the calibration routine will run.

Figure 5-8. Starting Calibrate TouchScreen from the Main Menu screen.

The TouchScreen Calibration screen is started by pressing “Calibrate TouchScreen” in the Main Menu screen.

Figure 5-9. TouchScreen Calibration.

Follow the instructions on the TouchScreen Calibration screen to calibrate.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

5.5

5.5 Technical Support

Technical Support

5.55.5

Technical SupportTechnical Support

For further assistance, contact Sutter Instrument Technical Support at:

(415) 883

(415) 883----0128

(415) 883(415) 883

0128 or

01280128

oror

info@sutter.com

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

APP E NDIX A.

APP E NDIX A. L IMIT E D WA RRAN TY

APP E NDIX A.APPEN D IX A .

LIM I TED W ARRA NTY

LIM I TED W ARRA NTYLIM I TED W ARRA NTY

Sutter Instrument Company, a division of Sutter Instrument Corporation, limits the warranty on this instrument to repair or replacement of defective components for one year after the date of shipment, provided the instrument has been operated in accordance with the instructions outlined in the instruction manual.

Abuse, misuse, or unauthorized repairs will void this warranty.

Limited warranty work will be performed only at the factory, and the cost of shipment both ways is to be borne by the user.

This instrument is designed to pull glass pipettes for use on animal tissues. It is not intended for use, nor should be used, in human experimentation, or applied to humans in any way.

The limited warranty is as stated above and no implied or inferred liability for direct or consequential damages is intended.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

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P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

APP E NDIX B.

APP E NDIX B. A C CES S ORIE S

APP E NDIX B.APPEN DIX B.

ACC E SSO R IES

ACC E SSO R IESACCES SORI E S

Filaments

Several sizes of of both Box and Trough filaments are available. Please refer to the Sutter Instrument Company’s web site (www.sutter.com) for a list of part numbers.

FPS

FPS Fire polishing spacer for P-1000 pullers

FPSFPS

GS Glass stop (

GSGS

CTS

CTS Ceramic tile for scoring glass (large tips 20-200 microns)

CTSCTS

BX10

BX10 Pipette storage box (holds 10) 4 3/4 x 3 5/8 x 3/4 inches

BX10BX10

BX20

BX20 Pipette storage box (holds 20) 7 x 3 5/8 x 3/4 inches

BX20BX20

installs on either puller bar)

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

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P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

APP E NDIX C.

APP E NDIX C. FU SE R EPLA C EME N T

APP E NDIX C.APPE N DIX C.

FUS E REP L ACE MENT

FUS E REP L ACE MENTFUSE R EPLA CEME N T

In the event that the controller fails to power up when the power switch is turned on, check the line power fuse to see if it has blown. The fuse is located in the fuse holder on the power entry module on the back of the controller. To remove the fuse holder first unplug the power cord from the power entry module. This will reveal a slot just under the edge of the fuse holder. Use a screwdriver to pry the holder straight out of the power entry module.

Power entry module. Fuse holder removed Fuse holder (spare fuse

Figure 5-10. Power entry module and fuse holder.

not shown)

The fuse that is readily visible in the fuse holder when you take it out is the one that is “active” when the holder is installed. A spare fuse is also stored within the fuse holder. Replace the active fuse with the spare and re-install the fuse holder and power cord. If the controller fails to power up with the new fuse installed, call Sutter Instrument technical support personnel for assistance.

Replace fuse only with the same type and rating:

Replace fuse only with the same type and rating: Replace fuse only with the same type and rating:

Type:

Type: Medium Time Delay (or Time Lag), 5 x 20 mm glas

Type:Type:

Rating:

Rating: T4A 250V (Time Delay, 4 Amps, 250 Volts)

Rating:Rating: Examples:

Examples: Bussmann S506

Examples:Examples:

Medium Time Delay (or Time Lag), 5 x 20 mm glass tube, IEC

Medium Time Delay (or Time Lag), 5 x 20 mm glasMedium Time Delay (or Time Lag), 5 x 20 mm glas compliant.

compliant.

compliant.compliant.

T4A 250V (Time Delay, 4 Amps, 250 Volts)

T4A 250V (Time Delay, 4 Amps, 250 Volts) T4A 250V (Time Delay, 4 Amps, 250 Volts) Bussmann S506----4444----R or Littelfuse 218

Bussmann S506Bussmann S506

R or Littelfuse 218 004.P (or 218

R or Littelfuse 218R or Littelfuse 218

004.P (or 218 004.HXP)

004.P (or 218004.P (or 218

s tube, IEC 60127

s tube, IECs tube, IEC

004.HXP)

004.HXP) 004.HXP)

60127----2, RoHS

2, RoHS

6012760127

2, RoHS 2, RoHS

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

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P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

APP E NDIX D.

APP E NDIX D. T E CHN I CAL SPEC I FICA T

APP E NDIX D.APP E NDIX D.

TEC H NICA L SP E CIF I CAT IONS

TEC H NICA L SP E CIF I CATTEC H NICA L SP E CIF I CAT

ION S

ION SI ONS

Dimensions (H x W x D):

Dimensions (H x W x D): 30 x 53 x 36 cm (12 x 21 x 14 in)

Dimensions (H x W x D):Dimensions (H x W x D):

30 x 53 x 36 cm (12 x 21 x 14 in)

30 x 53 x 36 cm (12 x 21 x 14 in)30 x 53 x 36 cm (12 x 21 x 14 in)

Weight:

Weight: 18.53 kg (40.85 lbs)

Weight:Weight:

18.53 kg (40.85 lbs)

18.53 kg (40.85 lbs)18.53 kg (40.85 lbs)

Electrical:

Electrical: Electrical:

Mains voltage

Mains voltage 100

Mains voltageMains voltage Power consumption

Power consumption 350W

Power consumptionPower consumption Power cord

Power cord 10A, 250V, with safety ground plug

Power cordPower cord Mains fuse (rear of cabinet)

Mains fuse (rear of cabinet) Time delay (or time lag) 5 x 20 mm glass tube.

Mains fuse (rear of cabinet)Mains fuse (rear of cabinet)

100 ---- 240 V, 50/60 Hz

240 V, 50/60 Hz

100 100

240 V, 50/60 Hz 240 V, 50/60 Hz

350W

350W 350W 10A, 250V, with safety ground plug

10A, 250V, with safety ground plug 10A, 250V, with safety ground plug Time delay (or time lag) 5 x 20 mm glass tube.

Time delay (or time lag) 5 x 20 mm glass tube. Time delay (or time lag) 5 x 20 mm glass tube. For specific fuse ratings, refer to

For specific fuse ratings, refer to Appendix C

For specific fuse ratings, refer to For specific fuse ratings, refer to

Appendix C

Appendix CAppendix C

....

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

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P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

APP E NDIX E.

APP E NDIX E. D R IERI TE M A TERI AL S A

APP E NDIX E.A PPE N DIX E.

IDENTITY: INDICATING DRIERITE DATE PREPARED 1-3-96 DESCRIPTION: 1/16" TO 1/4" BLUE GRANULES

DRI E RITE MAT ERIAL SA FETY DAT A SH E ET

DRI E RITE MAT ERIAL SADRI ERIT E MA T ERIA L SA

FETY DATA SH E ET

FETY DATA SH E ETFETY DATA SH E ET

SECTION I

MANUFACTURER’S NAME: W.A. HAMMOND DRIERITE CO. LTD. ADDRESS: P.0. BOX 460,

138 DAYTON AVE., XENIA, OH 45385

EMERGENCY PHONE NUMBER: (513) 376-2927 INFORMATION PHONE NUMBER: (513) 376-2927

SECTION II

IINNGGRREEDDIIEENNTTS

CHEMICAL IDENTITY % OSHA PEL ACGIH TLV UNITS C.A.S. # CALCIUM SULFATE 97 15 10 mg/M COBALT CHLORIDE 3 0.05* 0.05* mg/M3 7646-79-9

*(AS COBALT METAL)

HAZARDOUS MATERIALS IDENTIFICATION SYSTEM (HMIS) HEALTH FLAMMABILITY REACTIVITY PROTECTIVE EQUIPMENT 1 0 1 E

7778-18-9

SECTION III

PHYSICAL/CHEMICAL CHARACTERISTICS

SPECIFIC GRAVITY:(H20=1): 1.87 SOLUBILITY IN WATER: 0.25 GRAMS PER LITER MELTING POINT: 1450° C DECOMPOSES

APPEARANCE: BLUE GRANULES; NO ODOR

SECTION IV

FIRE AND EXPLOSION HAZARD DATA

FLASH POINT: NONE EXTINGUISHING MEDIA: NOT COMBUSTIBLE SPECIAL FIREFIGHTING PROCEDURES: NONE UNUSUAL FIRE AND EXPLOSION HAZARDS: NONE

SECTION V

REACTIVITY DATA

STABILITY: STABLE INCOMPATIBILITY (MATERIALS TO AVOID): STRONG ACIDS

HAZARDOUS DECOMPOSITION BYPRODUCTS: Cl2 @ 318°C; SO3 @ 1450°C HAZARDOUS POLYMERIZATION: WILL NOT OCCUR

SECTION VI

HEALTH HAZARD DATA

EYES: PARTICLES MAY CAUSE IRRITATION. SKIN: THIS MATERIAL IS NOT TOXIC. MAY DRY OR IRRITATE SKIN INHALATION: MAY CAUSE AN IRRITATION OF RESPIRATORY ORGANS OF SENSITIVE PERSONS

RESULTING IN THE OBSTRUCTION OF AIRWAYS WITH SHORTNESS OF BREATH.

INGESTION: MAY CAUSE VOMITING, DIARRHEA AND SENSATION OF WARMTH SIGNS AND SYMPTOMS OF OVER EXPOSURE: EYE, NOSE, THROAT, OR RESPIRATORY IRRITATION

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

CARCINOGENICITY OF INGREDIENTS: MATERIAL IARC NTP OSHA

CALCIUM SULFATE NOT LISTED NOT LISTED NOT LISTED COBALT CHLORIDE YES* NO NO *(COBALT & COBALT COMPOUNDS ARE CLASSIFIED AS GROUP 2B) MEDICAL CONDITIONS GENERALLY AGGRAVATED BY EXPOSURE:

PRE-EXISTING UPPER RESPIRATORY AND LUNG DISEASE SUCH AS, BUT NOT LIMITED TO, BRONCHITIS, EMPHYSEMA & ASTHMA

EMERGENCY AND FIRST AID PROCEDURES:

EYES: FLUSH WITH WATER DUST INHALATION: REMOVE TO FRESH AIR SKIN: WASH WITH WATER INGESTION: NONE KNOWN

SECTION VII

SPILL OR LEAK PROCEDURES

STEPS TO BE TAKEN IN CASE MATERIAL IS RELEASED OR SPILLED:

SWEEP OR VACUUM MATERIAL INTO APPROPRIATE WASTE CONTAINER FOR DISPOSAL. AVOID DUSTING CONDITIONS.

WASTE DISPOSAL METHOD: THIS MATERIAL CAN BE DISPOSED OF AS AN INERT SOLID WASTE IN AN APPROVED LAND FILL OR BY OTHER PROCEDURES ACCEPTABLE UNDER FEDERAL, STATE AND LOCAL REGULATIONS. PRECAUTIONS TO BE TAKEN IN HANDLING AND ST0RING: KEEP CONTAINER CLOSED STORE IN A COOL DRY PLACE AVOID GENERATING DUST

SECTION VIII

CONTROL MEASURES

RESPIRATORY PROTECTION: NIOSH/OSHA APPROVED FOR DUST VENTILATION: TO MEET TLV REQUIREMENTS EYES: SAFETY GLASSES OR GOGGLES OTHER PROTECTIVE EQUIPMENT: GLOVES OR PROTECTIVE CLOTHING ARE NOT USUALLY NECESSARY BUT

MAY BE DESIRABLE IN SPECIFIC WORK SITUATIONS.

SECTION IX

REFERENCES

U.S. DEPARTMENT OF LABOR – OSHA FORM APPROVED OMB NO.1218 -0072. OSHA HAZARD COMMUNICATION STANDARD 29 CFR 1910.1200 U. S. GYPSUM CO.

Although the information and recommendation set forth herein are presented in good faith and believed to be correct as of the date hereof, the W.A. Hammond DRIERITE Co. makes no representation as to the completeness or accuracy thereof. Information is supplied upon the condition that the person receiving same will make their own determination as to its suitability for their purpose prior to use. In no event will the W.A. Hammond DRIERITE Co. be responsible for damages of any nature whatsoever resulting from the use of or reliance upon information herein supplied. No representations or warranties, either expressed or implied, of merchantability, fitness for a particular purpose of or any other nature are made hereunder with respect to information or the product to which information refers.

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

IND E X

IND E XI NDE X

AAAA

accessories ..............................................................69

air

compressor ...........................................................3

Drierite canister...................................................4

jet...........................................................................3

jet adjustment ......................................................3

nozzle....................................................................3

pump.....................................................................3

solenoid.................................................................4

valve ......................................................................4

valve solenoid.......................................................4

air nozzle

alignment............................................................47

air valve ....................................................................4

air valve solenoid .....................................................4

angle plate ............................................................4, 5

aspiration pipettes.................................................24

BBBB

base ...........................................................................7

baseplate...................................................................7

box filaments..........................................................45

bumpers....................................................................6

CCCC

cable ..........................................................................6

adjustment................................................... 49, 50

cable pulley assembly

bumpers................................................................6

cable ......................................................................6

panels, L/R............................................................6

pull bars..............................................................47

puller bars.............................................................6

spring stops ..........................................................6

V-bearings ..........................................................47

cable pulley assembly-bearings ..............................6

cables................................................................ 49, 50

cables pulley assembly.............................................5

control functions

ramp test ............................................................29

cycle

definition ..................................................... 20, 22

DDDD

date..........................................................................20

default configuration...................................... 20, 31

delay

definition ..................................................... 21, 24

desiccant

regeneration .......................................................50

diagnostics..............................................................59

Air Pressure System..........................................60

Heat.....................................................................61

Pull......................................................................62

Velocity ...............................................................63

dimensions..............................................................73

of glass ..................................................................1

disclaimer ............................................................... iii

Drierite ...................................................................50

regeneration .......................................................50

Drierite canister.......................................................4

drierite material safety data sheet .......................75

FFFF

filament

adjustment............................................................5

filaments.................................................................43

box.......................................................................45

geometry...................................................... 44, 46

positioning................................................... 44, 45

reorder numbers......................................... 44, 46

replacement........................................................47

trough .................................................................44

fire polish................................................................23

fire polishing...........................................................42

front panel

rotary dial .............................................................2

fuse

holder..................................................................71

location ...............................................................71

replacement........................................................71

spare....................................................................71

fuses

mains

replacement.............................................. iii, 71

fuses, replacement

fuses, replacementfuses, replacement

mains

mains.............................................................iii, 71

mainsmains

GGGG

general information.................................................1

glass

dimensions............................................................1

glassware

glasswareglassware

alignment............................................................47

loading ................................................................13

precautions

precautions..........................................................iv

precautionsprecautions

specifications........................................................1

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

HHHH

heat

definition ............................................................21

heating assembly .....................................................4

angle plate ........................................................4, 5

eccentrics..............................................................5

heating filaments................................See filaments

holding type pipettes .............................................24

IIII

ID...............................................................................1

KKKK

keypad.......................................................................2

keys

numeric.................................................................2

LLLL

line power (mains)...................................................9

locked/unlocked......................................................20

MMMM

mains

mains.........................................................................9

mainsmains

fuses

fuses .............................................................. iii, 71

fusesfuses

memory...................................................................32

microinjection needle fabrication.........................33

microinjection pipettes..........................................23

microperfusion pipettes ........................................24

micropipette fabrication........................................33

NNNN

power entry module...............................................71

power switch ..........................................................10

pressure ..................................................................20

program

default configuration.........................................31

definition ..................................................... 20, 22

memory...............................................................32

parameters .........................................................32

selection..............................................................25

program line

definition ............................................................22

pull

definition ............................................................21

pull cycle.................................................................24

pull time..................................................................41

puller bar................................................................14

adjustment............................................................6

alignment..............................................................6

puller bars ................................................................6

pulleys

adjustment................................................... 49, 50

RRRR

ramp test ................................................................29

rotary dial .................................................................2

SSSS

safety warnings

safety warnings................................................ iii, 71

safety warningssafety warnings

mains fuse

mains fuse........................................................... iii

mains fusemains fuse

solenoid.....................................................................4

spring stops ..............................................................6

notes

user......................................................................80

OOOO

OD.............................................................................1

operation

operation.................................................................30

operationoperation

first time use ......................................................11

precautions

precautions..........................................................iv

precautionsprecautions

PPPP

panels, L/R................................................................6

parameter

adjustment..........................................................32

Patch pipette fabrication ......................................35

patch pipettes.........................................................23

pipette

aspiration............................................................24

holding (type).....................................................24

microperfusion...................................................24

patch ...................................................................23

pipette

microinjection ....................................................23

TTTT

technical specifications..........................................73

dimensions..........................................................73

electrical..............................................................73

mains fuse.......................................................73

mains voltage .................................................73

power consumption .......................................73

power cord......................................................73

weight .................................................................73

technical support .....................................................2

Technical Support .................................................66

time .........................................................................20

definition ..................................................... 21, 23

tip shapes................................................................53

tip size.....................................................................41

touchscreen

keypad...................................................................2

keys

numeric.............................................................2

Touchscreen Calibration.......................................65

troubleshooting......................................................53

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

controlling pipette tip shapes ...........................53

diagnostics..........................................................59

Air Pressure System......................................60

Heat.................................................................61

Pull..................................................................62

Velocity ...........................................................63

system operation/function problems................55

general operational........................................56

on-screen emergency reset messages...........56

on-screen error messages..............................57

startup ............................................................56

Technical Support .............................................66

Touchscreen Calibration...................................65

trough filaments ....................................................44

UUUU

unpacking.................................................................9

User Notes..............................................................26

VVVV

V-bearings ................................................................6

velocity

definition ............................................................21

special condition.................................................23

WWWW

warranty .................................................................67

weight .....................................................................73

write protection......................................................20

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

NOTES

NOTESNOTES

P-1000 FLAMING/BROWN MICROPIPETTE PULLER SYSTEM OPERATION MANUAL – REV. 1.06 (20110211)

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Frequently Asked Questions

User Manual