SUTTER INSTRUMENT P-97 Operation Manual

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OPERATION MANUAL

P----97

Flaming/Brown

Flaming/BrownFlaming/Brown

Micropipette

MicropipetteMicropipette

Puller

PullerPuller

Rev. 2.03 – DOM (20091301)

9797

P----97

Flaming/Brown Micropipette Puller

Flaming/Brown Micropipette PullerFlaming/Brown Micropipette Puller

9797

Operation Manual

(Rev. 2.03 – DOM (20091301))

Sutter Instrument Company

One Digital Drive Novato, CA 94949

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



P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

TABLE OF CONTENTS

TABLE OF CONTENTSTABLE OF CONTENTS

1. GENERAL INFORMATI

1. GENERAL INFORMATION

1. GENERAL INFORMATI1. GENERAL INFORMATI

1.1 T

ECHNICAL SUPPORT

1.2 W

HAT’S NEW ON THE

2. SAFETY WARNINGS A

2. SAFETY WARNINGS AND PRECAUTI

2. SAFETY WARNINGS A2. SAFETY WARNINGS A

2.1 M

AIN FUSE

2.2 A

VOIDING ELECTRICAL SHOCK OR FIRE INJURY

2.3 B

ACK INJURY PREVENTION

3. PRECAUTIONS

3. PRECAUTIONS ................................

3. PRECAUTIONS3. PRECAUTIONS

3.1 P

RECAUTIONS DURING OPERATION

3.2 ON H

4. GLASS CAPILLARY &

4. GLASS CAPILLARY & HEATING FILAMENT SP

4. GLASS CAPILLARY &4. GLASS CAPILLARY &

4.1 G

LASS CAPILLARY

4.2 H

EATING FILAMENT

.........................................................................................................................................3

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

ANDLING MICROPIPETTES

ON ................................

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

ONON

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

........................................................................................................................1

P-97?..............................................................................................................1

ND PRECAUTIONS

ND PRECAUTIND PRECAUTI

ONS................................

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

ONSONS

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

.............................................................................3

..............................................................................................................3

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

.................................................................................................5

.......................................................................................................5

HEATING FILAMENT SPECIFICATIONS

HEATING FILAMENT SP HEATING FILAMENT SP

..............................................................................................................................7

..........................................................................................................................7

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

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

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

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

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

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

ECIFICATIONS................................

ECIFICATIONSECIFICATIONS

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

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

...............................5555

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

................3333

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

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

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

..........7777

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

5. UNPACKING

5. UNPACKING................................

5. UNPACKING5. UNPACKING

6. SETTING UP

6. SETTING UP................................

6. SETTING UP6. SETTING UP

6.1 L

INE POWER (MAINS

7. FRONT PANEL

7. FRONT PANEL................................

7. FRONT PANEL7. FRONT PANEL

7.1 C

ONTROLS

7.2 D

ISPLAY

8. MECHANICAL DESCRI

8. MECHANICAL DESCRIPTION (PULLER ANATOM

8. MECHANICAL DESCRI8. MECHANICAL DESCRI

8.1 S

OME BASIC INFORMATION

8.2 A

IR COOLING SYSTEM

8.3 H

EATING ASSEMBLY

8.4 U

PPER PULLEY ASSEMBLY

8.5 C

ABINET

8.6 E

LECTRONICS

9. FIRST TIM

9. FIRST TIME USE

9. FIRST TIM9. FIRST TIM

10. PROGRAMS

10. PROGRAMS................................

10. PROGRAMS10. PROGRAMS

10.1 P

10.2 P

10.3 P

10.4 D

10.5 S

10.6 V

10.7 C

10.8 E

ROGRAM STRUCTURE ROGRAM LINE PULL CYCLE PARAMETERS ULL CYCLE

EFAULT CONFIGURATION

ELECTING A PROGRAM

IEWING A PROGRAM LEARING A PROGRAM FROM MEMORY DITING A PROGRAM

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

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

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

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

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

)......................................................................................................................11

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

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

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

..............................13

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

........................................................................................................................................13

............................................................................................................................................14

PTION (PULLER ANATOMY)

PTION (PULLER ANATOMPTION (PULLER ANATOM

Y)................................

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

Y)Y)

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

............................17

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

.............................................................................................................17

.....................................................................................................................17

........................................................................................................................18

..............................................................................................................20

...........................................................................................................................................21

...................................................................................................................................21

E USE ................................

E USEE USE

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

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

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

..........................23

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

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

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

..................................................................................................................25

................................................................................25

...................................................................................................................................26

...........................................................................................................27

................................................................................................................27

....................................................................................................................28

.......................................................................................28

.....................................................................................................................28

10.8.1 Entering a New Program......................................................................................................28

10.8.2 Editing an Existing Program................................................................................................29

....9999

........

..11

....

1111

1313

1717

2323

..25

....

2525

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

11. SOFTWARE CONTROL

11. SOFTWARE CONTROL FUNCTIONS

11. SOFTWARE CONTROL11. SOFTWARE CONTROL

11.1 RAMP TEST < 1 >......................................................................................................................31

11.2 CHANGE AIR PRESSURE < 2 >.............................................................................................32

11.3 AIR MODE < 3 >.........................................................................................................................33

11.4 AIR TIME AT START OF PULL < 4 > ...................................................................................33

11.5 AIR TIME AT END OF PULL < 5 >........................................................................................34

11.6 RESET TIME AND DATE < 6 > ..............................................................................................34

11.7 WRITE-PROTECT THE PROGRAM < 7 >............................................................................34

11.8 MEMORY TEST < 8 >...............................................................................................................34

12. PULLING PIPETTES

12. PULLING PIPETTES................................

12. PULLING PIPETTES12. PULLING PIPETTES

12.1 P

12.2 N

13. PARAMETER ADJUST

13. PARAMETER ADJUSTMENT

13. PARAMETER ADJUST13. PARAMETER ADJUST

13.1 G

13.2 M

13.3 P

13.4 S

13.5 T

13.6 F

ROCEDURES

OTES ON PROGRAM OPERATION

ENERAL INFORMATION

ICROPIPETTE/MICROINJECTION NEEDLE FABRICATION

13.2.1 HEAT ......................................................................................................................................37

13.2.2 PULL Strength.......................................................................................................................37

13.2.3 VELOCITY (Trip Point)........................................................................................................38

13.2.4 TIME Mode (Cooling)............................................................................................................38

13.2.5 Delay Mode (Cooling).............................................................................................................38

13.2.6 PRESSURE Adjustment.......................................................................................................39

13.2.7 Filament Width ......................................................................................................................39

ATCH PIPETTE FABRICATION

13.3.1 HEAT ......................................................................................................................................40

13.3.2 PULL Strength.......................................................................................................................40

13.3.3 VELOCITY (Trip Point)........................................................................................................40

13.3.4 TIME Mode (Cooling)............................................................................................................41

13.3.5 PRESSURE ............................................................................................................................41

TEP-BY-STEP PATCH PIPETTE PROGRAMMING

ECHNICAL TIPS FOR PULLING MICROPIPETTES

13.5.1 Regulating the Time it takes to pull a Sharp Pipette ........................................................43

13.5.1.1 Pipette Position..........................................................................................................................................................43

13.5.2 Regulating the Length and Tip Size of a Sharp Pipette....................................................43

13.5.2.1 Filament Width..........................................................................................................................................................43

13.5.2.2 Air Flow.......................................................................................................................................................................43

13.5.2.3 DELAY Mode of Active Cooling..............................................................................................................................44

IRE POLISHING

..................................................................................................................................35

FUNCTIONS................................

FUNCTIONS FUNCTIONS

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

MENT ................................

MENTMENT

............................................................................................................................44

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

...............................................................................................................37

.....................................................................................................40

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

................................................................................................36

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

.........................................................................41

.......................................................................43

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

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

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

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

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

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

.........................................................37

......................31

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

..................35

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

3131

3535

...37

......

3737

14. HEATING FILAMENT

14. HEATING FILAMENTSSSS ................................

14. HEATING FILAMENT14. HEATING FILAMENT

14.1 G

14.2 T

14.3 B

15. MAINTENANCE

15. MAINTENANCE................................

15. MAINTENANCE15. MAINTENANCE

ENERAL INFORMATION

ROUGH FILAMENT

14.2.1 Positioning..............................................................................................................................47

14.2.2 Geometry.................................................................................................................................47

OX FILAMENT

14.3.1 Positioning..............................................................................................................................49

14.3.2 Geometry.................................................................................................................................49

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

..............................................................................................................................48

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

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

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

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

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

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

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

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

..........................51

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

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

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

4747

5151

15.1 C

15.2 H

15.3 P

15.4 R

16. TROUBLESHOOTING

16. TROUBLESHOOTING................................

16. TROUBLESHOOTING16. TROUBLESHOOTING

16.1 C

16.2 C

16.3 T

LEANING

EATING FILAMENT REPLACEMENT

15.2.1 Systematic Filament Replacement.......................................................................................51

15.2.2 Air jet Position........................................................................................................................51

15.2.3 Positioning the Filament in Relation to the Glass Capillary ............................................51

ULLEY ADJUSTMENT EGENERATION OF DRIERITE GRANULES

ONTROLLING PIPETTE TIP SHAPES

16.1.1 Problem: What type of glass should be used?.....................................................................57

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

pipette be pulled?...............................................................................................................................57

16.1.3 Problem: Okay, but how can tips be made even smaller?.................................................57

16.1.4 Problem: How can the size of a patch-pipette be increased?.............................................58

16.1.5 Problem: Patch-pipette tips vary in size from pull to pull.................................................58

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

formed. How can this be done?.......................................................................................................58

16.1.7 Problem: The electrodes are bent. How can they be made to pull straight?..................58

16.1.8 Problem: One electrode is much longer than the other electrode....................................58

16.1.9 Problem: The shape and resistance of the pipette changes from pull to pull..................59

ONTROLLER PROBLEMS

16.2.1 Problem: The filament does not light up when pull is pressed........................................59

16.2.2 Problem: Display blank, fan not on, or locked up/overlap................................................60

16.2.3 Problem: Display shows a row of blocks.............................................................................60

16.2.4 Problem: Displayed program values are not correct.........................................................60

ECHNICAL SUPPORT

.......................................................................................................................................51

...........................................................................................51

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

...................................................................................54

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

..............................................................................................................59

....................................................................................................................60

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

...........................................................................................57

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

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

................57

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

5757

APPENDIX A. FUSE REP

APPENDIX A. FUSE REPLACEMENT

APPENDIX A. FUSE REPAPPENDIX A. FUSE REP

APPENDIX B. LIMITED

APPENDIX B. LIMITED WARR

APPENDIX B. LIMITED APPENDIX B. LIMITED

APPENDIX C. DISCLAIM

APPENDIX C. DISCLAIMER

APPENDIX C. DISCLAIMAPPENDIX C. DISCLAIM

APPENDIX D. TECHNICA

APPENDIX D. TECHNICAL SPECIFICATIONS

APPENDIX D. TECHNICAAPPENDIX D. TECHNICA

APPENDIX E. DRIERITE

APPENDIX E. DRIERITE MATERIAL SAFETY DAT

APPENDIX E. DRIERITEAPPENDIX E. DRIERITE

INDEX

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

INDEXINDEX

TABLE OF FIGURES

TABLE OF FIGURESTABLE OF FIGURES

Figure 6-1. P-97 Cabinet (rear view)......................................................................................................11

Figure 6-2. P-97 Cabinet (end view, left). ..............................................................................................11

Figure 6-3. Power connection..................................................................................................................11

Figure 7-1. P-97 Front Panel...................................................................................................................13

Figure 7-2. Program Display...................................................................................................................14

Figure 8-1. P-97 Base Plate (Top view, cover removed).......................................................................17

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

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

LACEMENTLACEMENT

WARRANTY

WARRWARR

ANTY................................

ANTYANTY

ER................................

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

ERER

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

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

L SPECIFICATIONSL SPECIFICATIONS

MATERIAL SAFETY DATA SHEET

MATERIAL SAFETY DAT MATERIAL SAFETY DAT

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

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

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

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

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

A SHEET................................

A SHEETA SHEET

...........................61

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

............................63

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

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

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

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

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

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

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

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

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

............65

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

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

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

..................69

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

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

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

6161

6363

6565

6767

6969

7171

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

Figure 8-2. Filament Block Assembly. ...................................................................................................18

Figure 8-3. Upper cable pulley assembly................................................................................................20

Figure 9-1. Left puller bar. ......................................................................................................................23

Figure 10-1. Power-On Display...............................................................................................................27

Figure 10-2. P-97 Program Display........................................................................................................27

Figure 10-3. Clear Program Display.......................................................................................................28

Figure 11-1. Access to Control Functions..............................................................................................31

Figure 12-1. Pull Cycle Report................................................................................................................35

Figure 12-2. Sample program..................................................................................................................36

Figure 12-3. Pull cycle report (multiple-loops)......................................................................................36

Figure 13-1. Side view of fire polishing spacer. .....................................................................................45

Figure 13-2. Fire Polish Spacer in Puller Bar. ......................................................................................45

Figure 14-1. End view of trough filament and glass.............................................................................47

Figure 14-2. End view of box filament. ..................................................................................................48

Figure 15-1. V-Groove bearings and puller bars...................................................................................51

Figure 15-2. Filament alignment............................................................................................................52

Figure 15-3. Micropipette shapes............................................................................................................53

Figure 15-4. Pulley Adjustment..............................................................................................................54

Figure 16-1. Power entry module. ..........................................................................................................61

Figure 16-2. Fuse holder..........................................................................................................................61

TABLE OF TABLES

TABLE OF TABLESTABLE OF TABLES

Table 10-1. Standard Factory Configuration.........................................................................................27

Table 11-1. Control Functions.................................................................................................................31

Table 11-2. Maximum heat and recommended starting values for different filament shapes........32

Table 14-1. Trough filament sizes. .........................................................................................................48

Table 14-2. Box filament sizes.................................................................................................................49

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

1. G E NERAL I NF ORMATI ON

GEN E RA L INFO RM ATION

1.1 .

GEN E RA L INFO RM ATIONGEN E RA L INFO RM ATION

The P-97 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 (as discussed in subsequent material).

The Model P-97 Flaming/Brown Micropipette Puller combines a proven pulling technology with programmability to produce a very versatile instrument. The pulling mechanism is derived from the P-77/P-80 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 multiplicity of pipettes on one device.

The P-97 is a ‘velocity sensing’ puller. This patented feature allows the puller to indirectly sense the viscosity of the glass, giving the P-97 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

www.sutter.com

to find a wide selection of glass

capillaries available for purchase.

1.1

1.1 Technical Support

Technical Support

1.11.1

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.

1.2

1.2 What’s New on the P

What’s New on the P----97?

1.21.2

What’s New on the PWhat’s New on the P

97?

97?97?

For users familiar with the P-80PC and/or P-87 pullers, the P-97 provides the advanced features incorporated into those instruments plus:



Humidity controlled chamber surrounding the heating filament. Glass is now pulled in a

Humidity controlled chamber surrounding the heating filament. Glass is now pulled in a Humidity controlled chamber surrounding the heating filament. Glass is now pulled in a constant humidity environment to minimize environmental effects on

constant humidity environment to minimize environmental effects on pipette

constant humidity environment to minimize environmental effects onconstant humidity environment to minimize environmental effects on reproducibility.

reproducibility.

reproducibility.reproducibility.



Vacuum fluorescent display for improved readability

Vacuum fluorescent display for improved readabilityVacuum fluorescent display for improved readability

pipette

pipette pipette



25% increase in available power

25% increase in available power25% increase in available power



Programmable air pressure

Programmable air pressureProgrammable air pressure



Time of pull displayed at the end of each pull sequence

Time of pull displayed at the end of each pull sequenceTime of pull displayed at the end of each pull sequence



Write protection to prevent programs from being corrupted

Write protection to prevent programs from being corruptedWrite protection to prevent programs from being corrupted

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)



Displa

Displayed date/time of last program edit

DisplaDispla



Increased memory capable of storing 100 programs

Increased memory capable of storing 100 programsIncreased memory capable of storing 100 programs



Switchable cooling mode to facilitate easy transfer of programs from both P

Switchable cooling mode to facilitate easy transfer of programs from both P----80PC and P

Switchable cooling mode to facilitate easy transfer of programs from both PSwitchable cooling mode to facilitate easy transfer of programs from both P 87 pullers

87 pullers

87 pullers87 pullers

yed date/time of last program edit

yed date/time of last program edityed date/time of last program edit

80PC and P----

80PC and P80PC and P

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

2. SA FE TY WARN I NG S AND

SAF E TY WARNI NG S AND PRECA UT IO NS

2.2 .

SAF E TY WARNI NG S AND SAF E TY WARNI NG S AND

2.1

2.1 Main Fuse

Main Fuse

2.12.1

Main FuseMain Fuse





Repl

Replace fuse only with the same type and rating:

ace fuse only with the same type and rating:

Repl Repl

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

Type:

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

Type:Type:

Rating:

Rating: 3A 250V (3 Amps, 250 Volts)

Rating:Rating:

Examples:

Examples: Bussmann GMC

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 A

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 Fuse

Fuse Replacement

Replacement for more details on fuse ratings and for instructions on how to change

FuseFuse

ReplacementReplacement

the fuse.

the fuse. the fuse.

2.2

2.2 Avoiding Electrical Shock or Fire Injury

Avoiding Electrical Shock or Fire Injury

2.22.2

Avoiding Electrical Shock or Fire InjuryAvoiding Electrical Shock or Fire Injury

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.

3A 250V (3 Amps, 250 Volts)

3A 250V (3 Amps, 250 Volts) 3A 250V (3 Amps, 250 Volts)

Bussmann GMC----3A or GMC

Bussmann GMCBussmann GMC

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

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

PRECA UTION S

PRECA UTION SPRE CA UTION S

3A or GMC----3333----R; or Littelfus

3A or GMC3A or GMC

R; or Littelfuse 239 003 or 239 003.P)

R; or LittelfusR; or Littelfus

60127----2, Sheet III) or

6012760127

e 239 003 or 239 003.P)

e 239 003 or 239 003.P)e 239 003 or 239 003.P)

2, Sheet III) or

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

Appendix A

Appendix AAppendix A



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 power source; appropriate

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

Use only a properly grounded power source; appropriateUse only a properly grounded power source; appropriate 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 occurs.

that an electrical hazard occurs.

that an electrical hazard occurs.that an electrical hazard occurs.



Do not disassemb

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

Do not disassembDo not disassemb

2.3

2.3 Back Injury Prevention

Back Injury Prevention

2.32.3

Back Injury PreventionBack Injury Prevention

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----97 Micropipette Puller weighs in excess of 18 kg (

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.

le the unit. Refer servicing to Sutter Instrument Company.

le the unit. Refer servicing to Sutter Instrument Company.le the unit. Refer servicing to Sutter Instrument Company.

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

97 Micropipette Puller weighs in excess of 18 kg (over 39 lb)

97 Micropipette Puller weighs in excess of 18 kg (97 Micropipette Puller weighs in excess of 18 kg (

ly rated for use with this

ly rated for use with this ly rated for use with this

over 39 lb)

over 39 lb) over 39 lb)

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

3. PR EC AU TI ONS

PRE C AU TI ONS

3.3 .

PRE C AU TI ONSPRE C AU TI ONS

3.1

3.1 Precautions during Operation

Precautions during Operation

3.13.1

Precautions during OperationPrecautions during Operation

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----97 using the indicated line voltage.

Operate the POperate the P



The P

The P----97 is designed for operation in a laboratory environment (pollution degree II) and

The PThe P at temperatures between 5°C

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

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



The P

The P----97 is designed for connection to a standard laboratory power outlet (overvoltage

The PThe P Category II) with main supply voltage fluct

Category II) with main supply voltage fluctuations not to exceed ± 10% of the normal

Category II) with main supply voltage fluctCategory II) with main supply voltage fluct voltage.

voltage.

voltage.voltage.



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 meters (6562 feet).

above 2000 meters (6562 feet).

above 2000 meters (6562 feet).above 2000 meters (6562 feet).



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

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 temperatureThis unit was designed to operate at maximum relative humidity of 80% for temperature 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.

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

97 is designed for operation in a laboratory environment (pollution degree II) and 97 is designed for operation in a laboratory environment (pollution degree II) and

97 is designed for connection to a standard laboratory power outlet (overvoltage

97 is designed for connection to a standard laboratory power outlet (overvoltage 97 is designed for connection to a standard laboratory power outlet (overvoltage

97 using the indicated line voltage.

97 using the indicated line voltage. 97 using the indicated line voltage.

40°C.

40°C. 40°C.

uations not to exceed ± 10% of the normal

uations not to exceed ± 10% of the normal uations not to exceed ± 10% of the normal

s s



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

Operate only in a location where there is a free flow of fresh air on all sides. The fan Operate only in a location where there is a free flow of fresh air on all sides. The fan draws air in through the vents on the sides and exhausts out both ends of the heat sink.

draws air in through the vents on the sides and exhausts out both ends of the heat sink.

draws air in through the vents on the sides and exhausts out both ends of the heat sink. draws air in through the vents on the sides and exhausts out both ends of the heat sink. NEVER ALL

NEVER ALLOW THE FREE FLOW OF AIR TO BE RESTRICTED.

NEVER ALLNEVER ALL



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.



Only use Sutter Instrument Company replacement heating filaments.

Only use Sutter Instrument Company replacement heating filaments.Only use Sutter Instrument Company replacement heating filaments.

3.2

3.2 On Handling Micropipettes

On Handling Micropipettes

3.23.2

On Handling MicropipettesOn 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 using t

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

The micropipettes created using tThe micropipettes created using t 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 (4.1

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

OW THE FREE FLOW OF AIR TO BE RESTRICTED.

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

his instrument are very sharp and relatively fragile.

his instrument are very sharp and relatively fragile. his instrument are very sharp and relatively fragile.

marked, spill----proof “sha

marked, spillmarked, spill

4.1).

4.14.1

).).

proof “sharps”

proof “shaproof “sha

rps”

rps” rps”

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

4. G L AS S CAPI LL A RY & HE

GLA S S CAPI L LA RY & H EATIN G FI LA MENT S PE CI

4.4 .

GLA S S CAPI L LA RY & H EGLA S S CAPI LL A RY & HE

4.1

4.1 Glass Capillary

Glass Capillary

4.14.1

Glass CapillaryGlass Capillary

The P-97 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-97 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

4.2

4.2 Heating Filament

Heating Filament

4.24.2

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-97.

www.sutter.com

ATIN G FILA ME NT SPECI FICAT I ON S

ATIN G FILA ME NT SPECIATIN G FILA ME NT SPECI

FICA TI ON S

FICA TI ON SFI CA TI ON S

ONLY USE SUTTER

ONLY USE SUTTER----SUPPLIED REPLACEMENT HEATING F

ONLY USE SUTTERONLY USE SUTTER INSTRUMENT!

INSTRUMENT!

INSTRUMENT!INSTRUMENT!

Systematic instructions for replacing the Heating Filament can be found in the Maintenance section of this manual.

SUPPLIED REPLACEMENT HEATING FILAMENTS IN THIS

SUPPLIED REPLACEMENT HEATING FSUPPLIED REPLACEMENT HEATING F

ILAMENTS IN THIS

ILAMENTS IN THIS ILAMENTS IN THIS

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

5. UN PA CKING

UNPA CKING

5.5 .

UNPA CKINGUN PA CKING

Make certain that you have received all of the following items in the P-97 shipping box:



P-97 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



Pipette Cookbook

The Model P-97 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,

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, IMPORTANT: Improper packaging is a form of abuse and, as such, 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 packingvoiding the warranty where shipping damage is sustained because of such packing

can be responsible for

can be responsible for can be responsible for

Unless specified otherwise, the P-97 is shipped equipped with, and configured for, a 2.5mm box heating filament.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

6. SE TT IN G UP

SET T IN G UP

6.6 .

SET T IN G UPSE TT IN G UP

6.1

6.1 Line Power (Mains)

Line Power (Mains)

6.16.1

Line Power (Mains)Line Power (Mains)

The power cord provided with the P-97 connects to the Power Entry Module located on the back of the unit (see diagram below). This Module also includes the Line Fuse.

Figure 6-1. P-97 Cabinet (rear view).

Make certain that the Power Switch located on the left end of the P-97 cabinet is turned OFF.

Figure 6-2. P-97 Cabinet (end view, left).

Plug the power cord provided with the P-97 into the Line Input socket on the Power Entry Module and then to a power source of the appropriate voltage and frequency.

Figure 6-3. Power connection.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

7. FR O NT PA N EL

FRON T PANE L

7.7 .

FRON T PANE LF RO NT PAN EL

Figure 7-1. P-97 Front Panel.

7.1

7.1 Controls

Controls

7.17.1

ControlsControls

Vacuum Fluorescent Display

Vacuum Fluorescent Display Displays program parameters.

Vacuum Fluorescent DisplayVacuum Fluorescent Display

Reset

Reset Re-initializes the controller.

ResetReset

Keypad

ypad Used to program parameter values and execute

KeKe

ypadypad

programs.

Keys:

Keys:Keys:

0000----9999 Used for choosing the desired program or control

function, entering numeric values when programming and to make YES/NO (1/0) decisions.

CLR

CLR Used to delete programs or numeric values entered into

CLRCLR

those programs. This key is also used to access the RAMP TEST.

ENTR

ENTR Used to enter new values.

ENTRENTR

NEXT Used to move to the next line in a program while

NEXTNEXT

editing.

LAST

LAST Used to move to the previous line in a program while

LASTLAST

editing.

PULL

STOP

PULL Initiates the execution of a program.

PULLPULL

STOP Aborts the execution of a program.

STOPSTOP

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

7.2

Write Protection (Off)

Last Date/Time edited

Pressure Setting

Program #

Program Line #

7.2 Display

Display

7.27.2

DisplayDisplay

The P-97 has a two line vacuum fluorescent display for easy viewing from any angle. The following figure demonstrates what you will see after you have selected a program. A brief description of each parameter is provided below.

UNLOCKED P=500 JAN 18, 1997 2:30 PM

UNLOCKED P=500 JAN 18, 1997 2:30 PMUNLOCKED P=500 JAN 18, 1997 2:30 PM 3 1 HEAT=330,PULL=150,VEL.= 80,TIME=200

3 1 HEAT=330,PULL=150,VEL.= 80,TIME=200

3 1 HEAT=330,PULL=150,VEL.= 80,TIME=2003 1 HEAT=330,PULL=150,VEL.= 80,TIME=200

Figure 7-2. Program Display.

PROGRAM

PROGRAMPROGRAM (0-99)

A program consists of one or more cycles when executed in sequence, will ‘pull’ the capillary

1111

which,

glass inserted in the instrument. A program can be up to 8 cycles in length

WRITE PROTECTION

WRITE PROTECTIONWRITE PROTECTION (Locked/Unlocked)

PRESSURE SETTING

PRESSURE SETTINGPRESSURE SETTING (Range 0-999)

DATE/TIME

DATE/TIME Reports the date and time of the last program edit.

DATE/TIMEDATE/TIME

HEAT

HEATHEAT (Range 0-999)

locked/unlocked"

}}}}Prevents program from being

edited when in Locked mode.

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

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.

PULL

PULLPULL (Range 0-255)

A CYCLE consists of four programmable parameters; H

sequence of events during execution of a cycle is described in the PROGRAMS SECTION of this manual.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

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.

EAT

, P

ULL

, V

ELOCITY

and either T

IME or DELAY

. A CYCLE is equivalent to one line of program code. The

VELOCITY

VELOCITYVELOCITY (Range 0-255)

TIME

TIMETIME (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 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 VELOCITY=0.

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 DISCUSSION OF THE SIGNIFICANCE OF TIME=0.

DELAY

DELAYDELAY (Range 0-255)

DELAY is a cooling mode, which 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.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

8. ME CH AN ICAL D ES CRIPTI

MEC H AN ICAL D ES CRIPTI ON (P U LLER A NA TOMY)

8.8 .

MEC H AN ICAL D ES CRIPTIMEC H AN ICAL D ES CRIPTI

8.1

8.1 Some Basic Information

Some Basic Information

8.18.1

Some Basic InformationSome Basic Information

This section presents a basic mechanical description of the P-97, 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.

8.2

8.2 Air Cooling System

Air Cooling System

8.28.2

Air Cooling SystemAir Cooling System

The Model P-97 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.

ON ( PU LLER A NA TOMY)

ON ( PU LLER A NA TOMY)ON ( PU LLER A NA TOMY)

Figure 8-1. P-97 Base Plate (Top view, cover removed).

Air Compressor

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

Air CompressorAir Compressor

to cool the filament and glass during the pull cycle.

Air Jet Nozzle

Air Jet Nozzle Directs the cooling air to the filament. The air jet nozzle

Air Jet NozzleAir Jet Nozzle

should be 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.

Air Valve Solenoid

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

Air Valve Solenoid Air Valve Solenoid

Drierite Canister

Drierite CanisterDrierite Canister (Reservoir Tank)

(Reservoir Tank)

(Reservoir Tank)(Reservoir Tank)

A canister filled with Drierite used to remove moisture from the air flowing between the pump and the valve solenoid. The dissected air is used to purge heat before and after a pull and to cool the air flowing to the filament and glass during the pull cycle.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

8.3

8.3 Heating Assembly

Heating Assembly

8.38.3

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 Chamber

Humidity Control ChamberHumidity Control Chamber Figure

Figure 8888----2222))))

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.

Filament Block Assembly

Filament Block AssemblyFilament Block Assembly ((((Figure

Figure 8888----2222))))

FigureFigure

Figure 8-2. Filament Block Assembly.

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

Brass Jaws. Note that

Brass JawsBrass Jaws these jaws may be moved up and down by loosening the screws (D)

(D) that secure them to the front of the filament

(D)(D) 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.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

Eccentrics and Angle Plate

Eccentrics and Angle PlateEccentrics and Angle Plate ((((Figure

Figure 8888----2222))))

FigureFigure

Cover Plate

Cover PlateCover Plate ((((Figure

Figure 8888----2222))))

FigureFigure

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

B’

B’ are the corresponding locking screws. By turning the

B’B’

A’ and

A’A’

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

(B)(B) adjust the position of the 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.

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 Jaws constitute the ‘coarse adjustments’

NOTE: The movements of the Cover Plate and the Jaws constitute the ‘coarse adjustments’ NOTE: The movements of the Cover Plate and the Jaws constitute the ‘coarse adjustments’ of filament position, while the eccentric screws a

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

of filament position, while the eccentric screws aof filament position, while the eccentric screws a

llow ‘fine adjustments’.

llow ‘fine adjustments’.llow ‘fine adjustments’.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

8.4

8.4 Upper Pulley Assembly

Upper Pulley Assembly

8.48.4

Upper Pulley AssemblyUpper Pulley Assembly

This assembly guides the Pulling Cables 8888----3333)))) 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 8888----3333)))), and contains a large eccentric adjustment screw (J in

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

Figure 8888----3333)))) from the Puller Bars

FigureFigure

(J in Figure

Figure 8888----3333)))). This eccentric screw

(J in (J in

FigureFigure

Puller Bars (G in

Puller BarsPuller Bars

(J’ in Figure

(J’ in (J’ in

(G in Figure

Figure

(G in (G in

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.

Figure 8-3. Upper cable pulley assembly.

Panels, Left And Right

Panels, Left And RightPanels, Left And Right (K in

(K in Figure

Figure 8888----3333))))

(K in (K in

FigureFigure

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 8888----3333))))

(N in (N in

FigureFigure

Spring Stops

Spring StopsSpring Stops (P in

(P in Figure

Figure 8888----3333)

(P in (P in

FigureFigure

Puller Bars

Puller BarsPuller Bars (G in

(G in Figure

Figure 8888----3333))))

(G in (G in

FigureFigure

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

)

) )

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) shallow groove (I)

(I) at the end of the puller bar, and

(I)(I)

(H) holds the cable in a

(H)(H)

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

VVVV---- Bearings

Bearings

Bearings Bearings

(S in

(S in Figure

Figure 8888----3333))))

(S in (S in

FigureFigure

Pull Cable

Pull CablePull Cable (T in

(T in Figure

Figure 8888----3333))))

(T in (T in

FigureFigure

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 Section). Note

NEVERNEVER 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.

This cable transmits the pulling force of the solenoid to the Puller Bars via the Upper (F)

(F) and Lower Pulley

(F)(F) Assemblies. It is made of 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.

8.5

8.5 Cabinet

Cabinet

8.58.5

CabinetCabinet

Baseplate

Baseplate The top think metal plate on which the mechanical

BaseplateBaseplate

assemblies are mounted.

Base

Base The Base includes the cabinet to which the top

BaseBase

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

8.6

8.6 Electronics

Electronics

8.68.6

ElectronicsElectronics

The P-97 micropipette puller is controlled by a Z-80 microprocessor. Three digital-to-analog (D-A) converters control the HEAT, PULL and VELOCITY values. The HEAT power supply is a precision constant current switching unit, which will vary less than 10 milliamperes with a plus or minus 10% change in the AC line current. 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-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

9. FI RS T TIME U SE

FIR S T TIME U SE

9.9 .

FIR S T TIME U SEF IR ST T IME U S E

While we realize that most new users of the P-97 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 because the user did not 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.

1. Make certain that the P-97 is plugged into the power outlet of the correct voltage and frequency.

2. Remove the rubber bands holding the puller bars together.

3. Turn Power switch on left side of cabinet ON

4. Press < 0 > followed flashing on the HEAT

< 0 > followed by < ENTR >

< 0 > followed< 0 > followed

HEAT parameter.

HEATHEAT

< ENTR > on the keypad to view Program 0. The cursor will be

< ENTR > < ENTR >

ON.

ONON

5. Inspect the parameter values displayed for Program 0. Program 0 should display the factory-installed values listed on the enclosed program sheet. If the values vary, jot down the values displayed and then re-enter the program sheet values.

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 units.

more than 10 units.

more than 10 units. more than 10 units.

6. Load a piece of the supplied sample glass into puller as follows:

Figure 9-1. Left puller bar.

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.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

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.

7. Press the < PULL >

< PULL > key on the keypad. The heating filament should turn on and the

< PULL >< PULL >

glass should separate in less than 10 seconds.

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

Unless otherwise stated on your program sheet, Program 0 is factory pre-programmed to pull a micropipette (tip diameter less than 0.1 mm) from 1.0mm x 0.5mm borosilicate glass. Program 1 is factory pre-programmed to pull a patch type pipette with a tip diameter of about 2mm. To try the patch pipette program (#1):

1. Press RESET to exit Program 0

2. Press < 1 >

< 1 > followed by < ENTR >

< 1 > < 1 >

< ENTR > to enter into Program 1.

< ENTR > < ENTR >

3. Repeat Steps 4-7 above.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

10.

10. PR OG RA MS

PROG RA MS

10.10 .

PROG RA MSPROGR A MS

10.1

10.1 Program Structure

Program Structure

10.110.1

Program StructureProgram Structure

The resulting size and shape of a micropipette made using the P-97 is determined by the parameter values that are programmed by the user. One hundred (100) separate programs can be saved for future use. 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 be up to 8 lines/cycles in length.

Cyc

Cycle

le A Cycle

CycCyc

lele

Cycle consists of five programmable parameters; Heat

CycleCycle either Time Program code.

Time or Delay,

TimeTime

Delay, and Pressure

Delay, Delay,

Pressure. A Cycle

Pressure Pressure

Cycle is equivalent to one line of

CycleCycle

Heat, Pull

Pull, Velocity

HeatHeat

PullPull

Velocity,

VelocityVelocity

10.2

10.2 Program Line Pull Cycle Parameters

Program Line Pull Cycle Parameters

10.210.2

Program Line Pull Cycle ParametersProgram Line Pull Cycle Parameters

Each of the four programmable parameters in a Cycle 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.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

VELOCITY

VELOCITYVELOCITY (Range 0-255)

TIME

TIMETIME (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. ..

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.

DELAY

DELAYDELAY (Range 0-255)

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 = 0 Special Condition: DELAY = 0 Special Condition: DELAY = 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.

10.3

10.3 Pull Cycle

Pull Cycle

10.310.3

Pull CyclePull Cycle

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

1. The heat turns on.

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

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

3. 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.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

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.

10.4

10.4 Default Configuration

Default Configuration

10.410.4

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 10-1. Standard Factory Configuration.

Heating Filament Installed

Heating Filament Installed 3mm Trough (Catalog #FT330B

Heating Filament InstalledHeating Filament Installed

3mm Trough (Catalog #FT330B))))

3mm Trough (Catalog #FT330B3mm Trough (Catalog #FT330B

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 (tip diameter less than 0.1 µm 1 -Patch type pipette

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, in general, 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 value for the 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 value for the the heating filament can be destroyed by an excessive value for the

through this section of the manual and have run the Ramp Test described herein.

10.5

10.5 Selecting a Program

Selecting a Program

10.510.5

Selecting a ProgramSelecting a Program

< 0 to 99 >

< 0 to 99 >< 0 to 99 > After applying power to the instrument, a “power on” reset will occur and the display will appear as follows:

WHICH PROGRAM DO YOU WANT? (0-99)

WHICH PROGRAM DO YOU WANT? (0-99)WHICH PROGRAM DO YOU WANT? (0-99) COPYRIGHT SUTTER INSTRUMENT CORP. 1992

Figure 10-1. Power-On Display.

The P-97 has the capacity to store 100 programs (0 - 99). On the keypad, press < 0 > followed by < ENTR program you wish to execute. The display will appear as shown below in Figure

ENTR > (factory installed micropipette program) or the number of another

ENTRENTR

Figure 10

FigureFigure

10----2222

1010 (numerical values may vary). Please refer to the FRONT PANEL chapter and the beginning of this chapter for a full description of the PARAMETERS shown.

UNLOCKED P=500 JAN 18, 1997 2:30 PM

UNLOCKED P=500 JAN 18, 1997 2:30 PMUNLOCKED P=500 JAN 18, 1997 2:30 PM 3 1 HEAT=330,PULL=150,VEL.= 80,TIME=200

3 1 HEAT=330,PULL=150,VEL.= 80,TIME=200

3 1 HEAT=330,PULL=150,VEL.= 80,TIME=2003 1 HEAT=330,PULL=150,VEL.= 80,TIME=200

Figure 10-2. P-97 Program Display.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

To select a different program, Press < RESET

press the number of the desired program (0 to 99) followed by < ENTR

10.6

10.6 Viewing a Program

Viewing a Program

10.610.6

Viewing a ProgramViewing a Program

< NEXT > / < LAST >

< NEXT > / < LAST >< NEXT > / < LAST > The front panel display limits you to viewing only two adjacent lines of a PROGRAM at one time. When you first select a PROGRAM, the display above will appear. The top line of every program is a header line, which displays the PRESSURE setting, the WRITE PROTECT STATUS and the LAST EDIT DATE. The second line of the display is Line 1 of program.

To view additional lines of the program

Press the < NEXT

RESET > to bring up the sign-on prompt then

RESETRESET

ENTR >.

ENTRENTR

NEXT > key

NEXTNEXT

To scroll to previous lines Press the < LAST

10.7

10.7 Clearing a Program from Memory

Clearing a Program from Memory

10.710.7

Clearing a Program from MemoryClearing a Program from Memory

< CLR >

< CLR >< CLR > To clear the values of a particular program from memory you can utilize the CLEAR function.

Pressing the < CLR >

Entering < 1111 > will clear the PROGRAM from the line the cursor is on to the end of the PROGRAM. If the cursor is on Line 1, the entire program will be cleared from memory. If the cursor is on Line 2, Line 1 will be preserved, and lines 2-8 will be cleared.

10.8

10.8 Editing a Program

Editing a Program

10.810.8

Editing a ProgramEditing a Program

Select a program number (e.g. 10). If there are already values entered, make sure that this program was not entered by another user of the puller. Unused program areas are usually cleared before a puller is shipped, but occasionally test program values are inadvertently left in memory. We recommend that unused programs be cleared before proceeding.

< CLR > key gives you the following message:

< CLR >< CLR >

UNLOCKED P=500 JAN 18, 1997 2:30 PM

UNLOCKED P=500 JAN 18, 1997 2:30 PMUNLOCKED P=500 JAN 18, 1997 2:30 PM 3 1 HEAT=330,PULL=150,VEL.= 80,TIME=200

3 1 HEAT=330,PULL=150,VEL.= 80,TIME=200

3 1 HEAT=330,PULL=150,VEL.= 80,TIME=2003 1 HEAT=330,PULL=150,VEL.= 80,TIME=200

Figure 10-3. Clear Program Display.

LAST > key.

LASTLAST

10.8.1

10.8.1 Entering a New Program

10.8.110.8.1 After selecting or clearing a program area, the cursor will be blinking at the HEAT

parameter on Line 1. To familiarize yourself with program entry, enter the following sample micropipette program:

Entering a New Program

Entering a New ProgramEntering a New Program

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

HEAT

HEAT PULL

HEATHEAT

300 150 100 150

PULL VELOCITY

PULLPULL

VELOCITY TIME

VELOCITYVELOCITY

TIME

TIMETIME

Press the series of numbers < 300 > to enter the HEAT value of 300. The cursor will automatically tab over to the PULL parameter.

Enter < 150 > for PULL. The cursor will tab over to the VELOCITY parameter.

Enter < 90 > for VELOCITY. The cursor will remain there, blinking. Note that when less than three digits are entered, the cursor will not automatically tab to the next field.

than three digits are entered, the cursor will not automatically tab to the next field. You

than three digits are entered, the cursor will not automatically tab to the next field.than three digits are entered, the cursor will not automatically tab to the next field. must press < ENTR

ENTR > to enter the value and tab to the next field.

ENTRENTR

Note that when less

Note that when less Note that when less

Enter < 150 > for TIME. The cursor will tab to the next field, which is HEAT in Line 2.

10.8.2

10.8.2 Editing an Existing Program

10.8.210.8.2

Editing an Existing Program

Editing an Existing ProgramEditing an Existing Program

To edit the value of a parameter(s) in a particular line, the cursor must be blinking in the field you want to edit. If necessary, press < NEXT > want to change. Press < ENTR >

< ENTR > to tab the cursor to the field you want to change, and

< ENTR >< ENTR >

< NEXT > or < LAST >

< NEXT >< NEXT >

< LAST > to scroll to the line you

< LAST >< LAST >

enter the new value. In our example above, to change the value of VELOCITY in Line 1 you first press < LAST >

< LAST > to scroll up to Line 1. Then press < ENTR >

< LAST >< LAST >

< ENTR > two times to tab over to

< ENTR >< ENTR > the VELOCITY field. Note that the values for HEAT and PULL remain unchanged. Enter a new value (e.g. 90); if the value is two digits, you must press < ENTR > The cursor will be positioned on Line 1 in the TIME field. To move to Line 2, press < ENTR

< ENTR > to enter the value.

< ENTR > < ENTR >

< ENTR

< ENTR < ENTR

>>>> to “tab” over to the HEAT field in Line 2.

The program values above are only given to demonstrate data entry procedures. Useful program values to pull the pipette that you want will be discussed in the next chapter. In general, inappropriate settings will only affect your ability to control the geometry of the glass micropipette you are trying to fabricate. However, the heating element can be des

destroyed by an excessive value for the HEAT parameter

troyed by an excessive value for the HEAT parameter. Do not use HEAT settings greater

desdes

troyed by an excessive value for the HEAT parametertroyed by an excessive value for the HEAT parameter

than those found in programs 0 and 1 until you have run the RAMP TEST

than those found in programs 0 and 1 until you have run the RAMP TEST described in the

than those found in programs 0 and 1 until you have run the RAMP TEST than those found in programs 0 and 1 until you have run the RAMP TEST

the heating element can be

the heating element can be the heating element can be

Do not use HEAT settings greater

Do not use HEAT settings greater Do not use HEAT settings greater

next section.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

11.

11. SO FT WA RE CON TR OL FUN

SOF T WA RE CON TR OL F UNCT IO NS

11.11 .

SOF T WA RE CON TR OL F UNSOF T WA RE CON TR OL F UN

CTIO N S

CTIO N SC TI ON S

The P-97 has several unique software CONTROL FUNCTIONS that allow you to, among other things, run SELF-TEST procedures, set up some of the PULL CONDITIONS and WRITE-PROTECT your program. These CONTROL FUNCTIONS are accessed through the < CLR

CLR > key. Pressing < CLR

CLRCLR

CLR > gives you the following message:

CLRCLR

DO YOU WISH TO CLEAR ALL VALUES FROM THE

DO YOU WISH TO CLEAR ALL VALUES FROM THEDO YOU WISH TO CLEAR ALL VALUES FROM THE PRESENT CYCLE TO THE END? NO=

PRESENT CYCLE TO THE END? NO=0 YES=1

PRESENT CYCLE TO THE END? NO=PRESENT CYCLE TO THE END? NO=

0 YES=1

0 YES=10 YES=1

Figure 11-1. Access to Control Functions.

Entering < 0 >

< 0 > will provide you with access to the following P-97 CONTROL FUNCTIONS:

< 0 >< 0 >

Table 11-1. Control Functions.

Function

Function Function

####

Function Name

Function Name Description

Function NameFunction Name

Description

DescriptionDescription

1 RAMP TEST Run the Ramp Test

2 CHANGE AIR PRESSURE Set the air pressure for a program

3 AIR MODE Select the active cooling mode

4 AIR TIME AT START OF PULL

5 AIR TIME AT END OF PULL Purge chamber at end of pull

6 RESET TIME AND DATE Set the clock

8 MEMORY TEST Test the integrity of the RAM

WRITE-PROTECT THE PROGRAM

Dehumidify the heating chamber prior to pulling.

Lock or unlock the write protection

The eight CONTROL FUNCTIONS will scroll along the bottom line of the display. Select the desired CONTROL FUNCTION by pressing the corresponding numeric key. Each CONTROL FUNCTION is more completely described below.

11.1

11.1 RAMP TEST < 1 >

RAMP TEST < 1 >

11.111.1

RAMP TEST < 1 > RAMP TEST < 1 >

The HEAT value required to melt your glass is based on the characteristics of the heating filament that is installed and the 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.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

To run a RAMP TEST:

1. Go into any program. For example, Press <1>

2. Press <CLR>

3. Press <0>

4. Select <1>

5. Install glass and press <PULL>

<CLR>

<0> to not clear parameters

<0><0>

<1> for RAMP TEST

<1><1>

<PULL>

<1> and then <ENTR>

<1><1>

<ENTR> to select Program 1

When executed, events in the RAMP TEST take place as follows:

1. The puller increments the HEAT at the rate of 650 milliamps per second, and the

filament, after 1-2 minutes, begins to get hot and glows orange in color.

2. The puller bars begin to move apart as the HEAT output begins to soften the glass.

3. The HEAT is turned off when a certain velocity (the value of which is stored in ROM) is

achieved.

4. The RAMP TEST value shown on the display is the HEAT value that was required to

reach the factory-set RAMP TEST velocity.

To interrupt the RAMP TEST or reset the display press RESET.

Recommendations

RecommendationsRecommendations

Table 11-2. Maximum heat and recommended starting values for different filament shapes.

Filament

Filament Maximum/Recommended

FilamentFilament

Maximum/Recommended Value(s)

Maximum/RecommendedMaximum/Recommended

Value(s)

Value(s)Value(s)

Maximum program HEAT value(s) Ramp value + 35 units. Trough

Recommended starting value Ramp value+ 15 units.

Maximum Program HEAT value(s) Ramp value + 30 units. Box

Recommended starting value Ramp value.

Additional guidelines for setting program HEAT values relative to RAMP TEST values are given in the Programming Section of this manual. HEAT sett damage or burn out the filament.

damage or burn out the filament.

damage or burn out the filament.damage or burn out the filament.

11.2

11.2 CHANGE AIR PRESSURE < 2 >

CHANGE AIR PRESSURE < 2 >

11.211.2

CHANGE AIR PRESSURE < 2 >CHANGE AIR PRESSURE < 2 >

HEAT settings over RAMP +35 can

HEAT settHEAT sett

ings over RAMP +35 can

ings over RAMP +35 can ings over RAMP +35 can

This control sets the pressure generated by the air compressor during the active cooling phase of the pull cycle. Each time a pull is started the air pressure is corrected before the filament turns on. A value must be entered each time this function is accessed. Changes of less than 10 units will not be noticeable. Air pressure is reported on the program header line.

Range

Range 1 to 999

RangeRange

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

phase. The onset of the hard pull is set at 40ms. One unit of

Default

Default 500

DefaultDefault

Recommendations

RecommendationsRecommendations

For thick walled glass 500

For thin walled glass 300

11.3

11.3 AIR MODE < 3 >

AIR MODE < 3 >

11.311.3

AIR MODE < 3 >AIR MODE < 3 >

Select between one of two modes of cooling during the active cooling phase of the pull cycle. The mode is reported in the fourth field of each program line.

TIME MODE

TIME MODE Controls the length of time the gas is on during the cooling

TIME MODETIME MODE

TIME is equal to 1/2 millisecond (e.g., at a setting of 150 the cooling air will be on for 75ms).

DELAY MODE

DELAY MODE Controls the onset of the hard pull. The gas is turned on for

DELAY MODEDELAY MODE

300 ms during the cooling phase. One unit of DELAY is equal to 1/2 millisecond (e.g. a setting of 80 will delay the onset of the hard pull by 40 ms).

Default

Default TIME MODE

DefaultDefault

Recommendations

RecommendationsRecommendations

TIME MODE

TIME MODETIME MODE

Use the TIME MODE for: Patch pipette programs

Glass <1.2mm O.D.

Transfer of model P-87 puller programs to P-97

Use the DELAY MODE for: Single cycle programs

Glass >1.2mm O.D.

Transfer of model P80-PC puller programs to P-97.

11.4

11.4 AIR TIME AT START OF PULL < 4 >

AIR TIME AT START OF PULL < 4 >

11.411.4

AIR TIME AT START OF PULL < 4 >AIR TIME AT START OF PULL < 4 >

Controls the length of time the humidity control chamber is purged with dehumidified air prior to execution of the pull sequence. The intent of the control is to minimize and stabilize the chamber humidity during the pull sequence. Units are in seconds.

Range

Range 1 to 255 seconds

RangeRange

Default

Default 5 seconds

DefaultDefault

Recommendations

RecommendationsRecommendations

For humid environments 20 to 30 seconds

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

11.5

11.5 AIR TIME AT END OF PULL < 5 >

AIR TIME AT END OF PULL < 5 >

11.511.5

AIR TIME AT END OF PULL < 5 >AIR TIME AT END OF PULL < 5 >

Controls the length of time the chamber is cooled after completion of the pull and the length of time the end of pull information is displayed. Units are in seconds. The time you enter is actually an offset added to an automatic 3 second chamber cooling cycle [e.g. A setting of 5 would result with 8 seconds of chamber cooling].

Range

Range 0 to 255 seconds

RangeRange

Default

Default 5 seconds

DefaultDefault

Recommendations

RecommendationsRecommendations

For single cycle programs 5 seconds

For multicycle programs (to allow additional cooling of the heating clamp assembly)

11.6

11.6 RESET TIME AND DATE < 6 >

RESET TIME AND DATE < 6 >

11.611.6

RESET TIME AND DATE < 6 >RESET TIME AND DATE < 6 >

This function allows you to set the system clock. The clock is used to establish the date and time of the last program edit as displayed on the program header line.

11.7

11.7 WRITE

WRITE----PROTECT THE PROGRAM < 7 >

11.711.7

WRITEWRITE

Activate (lock) or inactivate (unlock) the WRITE PROTECTION mode. Unlocked mode (0) allows program to be edited. Locked mode (1) prevents program from being edited. The write-protect status is reported on the program header line.

Default

Default Unlocked

DefaultDefault

11.8

11.8 MEMORY TEST < 8 >

MEMORY TEST < 8 >

11.811.8

MEMORY TEST < 8 >MEMORY TEST < 8 >

Performs a non-destructive test of the RAM. Press RESET to reset the system after performing this test.

PROTECT THE PROGRAM < 7 >

PROTECT THE PROGRAM < 7 >PROTECT THE PROGRAM < 7 >

25 seconds

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

12.

12. PU L LING PI PE TT ES

PUL L IN G PIPE TT ES

12.12 .

PUL L IN G PIPE TT ESPUL L IN G PIPE TT ES

12.1

12.1 Procedures

Procedures

12.112.1

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 note the Ramp value reported. The HEAT settings in the

TESTTEST factory-installed programs are typically set at Ramp value plus 15 units. If your Ramp test value differs from the factory Ramp value, adjust the HEAT in your programs to your Ramp value plus 15 units.

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 acquaint yourself with the pulling process.

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

2. Press < 0 > followed by < ENTR > on the keypad to view Program 0.

run the RAMP

run the RAMP run the RAMP

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 plus 15 units.

4. Press the < PULL > key on the keypad. 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 on, similar to the following:

THE PROGRAM LOOPED 01 TIMES. THE LAST

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

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

A unique feature of the P-97 is its capability to loop through a program. This is demonstrated with the multicycle Program 1, which is factory pre-programmed to pull a patch type pipette with a tip diameter of about 2mm. Press RESET to exit Program 0, and then press < 1111 > followed by < ENTR should read similar to the following:

THE PROGRAM LOOPED 01 TIMES. THE LASTTHE PROGRAM LOOPED 01 TIMES. THE LAST LINE USED WAS 01, HEAT ON SEC. = 6.05

LINE USED WAS 01, HEAT ON SEC. = 6.05

LINE USED WAS 01, HEAT ON SEC. = 6.05LINE USED WAS 01, HEAT ON SEC. = 6.05

Figure 12-1. Pull Cycle Report.

ENTR > to enter into Program 1. The display for Program 1

ENTRENTR

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

UNLOCKED P=500 JAN 18, 1997 2:35 PM

UNLOCKED P=500 JAN 18, 1997 2:35 PMUNLOCKED P=500 JAN 18, 1997 2:35 PM 1 1 HEAT=285,PULL= 0,VEL.= 50,TIME=150

1 1 HEAT=285,PULL= 0,VEL.= 50,TIME=150

1 1 HEAT=285,PULL= 0,VEL.= 50,TIME=1501 1 HEAT=285,PULL= 0,VEL.= 50,TIME=150

Figure 12-2. Sample program.

Adjust the HEAT in the program to the Ramp value. Load glass into the puller and press < PULL >

PULL >. The heating filament should cycle on and off repeatedly. When the pull is

PULL >PULL > complete, the display should read similar to the following:

THE PROGRAM LOOPED 04 TIMES. THE LAST

After the heat turns on in line 01, the glass heats up and draws apart until it reaches a VELOCITY of 50, 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 01 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.

12.2

12.2 Notes on Program Operation

Notes on Program Operation

12.212.2

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 PROGRAM LOOPED 04 TIMES. THE LASTTHE PROGRAM LOOPED 04 TIMES. THE LAST LIN

LINE USED WAS 01, HEAT ON SEC. = 14.7

E USED WAS 01, HEAT ON SEC. = 14.7

LINLIN

E USED WAS 01, HEAT ON SEC. = 14.7E USED WAS 01, HEAT ON SEC. = 14.7

Figure 12-3. Pull cycle report (multiple-loops)..

< <

All programs entered into memory (maximum of 100) remain there even after the power is turned off or the RESET switch is toggled. A special memory ‘chip’ that carries its own battery back up will retain stored information for as long as ten years without power being applied to the instrument. Miracle that this is, it is strongly suggested that you keep a written record of your programs in case of unexpected difficulties.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

13.

13. PA RA METER A DJUST M EN T

PARA ME TER AD J USTMEN T

13.13 .

PARA ME TER AD J USTMEN TPARA ME TER AD J USTMEN T

13.1

13.1 General Information

General Information

13.113.1

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 fabrication Technical Tips

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

Technical TipsTechnical Tips

Micropipette/microinjection needle fabrication, Patch pipette fabricat

Micropipette/microinjection needle fabricationMicropipette/microinjection needle fabrication

Patch pipette fabrication

Patch pipette fabricatPatch pipette fabricat

ion and

ionion

you read through 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

13.2

13.2 Micropipette/Microinjection Needle Fabrication

Micropipette/Microinjection Needle Fabrication

13.213.2

Micropipette/Microinjection Needle FabricationMicropipette/Microinjection Needle Fabrication

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

TIMETIME

Consider the following programs using a 3mm-trough filament:

Micropipette Program using 1.0mm x 0.5mm borosilicate tubing

HEAT

HEAT PULL

HEATHEAT

Ramp + 16 150 100 150 500

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 + 16 60 80 200 300

PULL VELOCITY

PULLPULL

VELOCITY TIME

VELOCITYVELOCITY

TIME PRESSURE

TIMETIME

PRESSURE

PRESSUREPRESSURE

13.2.1

13.2.1 HEAT

13.2.113.2.1

HEAT

HEATHEAT

The HEAT setting will affect the length and tip size of the pipette. Generally speaking, higher HEAT settings tend to give longer and finer tips. For trough filaments, the recommended starting HEAT value is the ramp test value plus 15 units. 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 > key is pressed. If the time is

< PULL >< PULL > longer than eight seconds, 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.

13.2.2

13.2.2 PULL Strength

13.2.213.2.2

PULL Strength

PULL StrengthPULL 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

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

micropipettes. The PULL strength can be set to any value desired with no danger of damaging the instrument.

13.2.3

13.2.3 VELOCITY (Trip Point)

13.2.313.2.3

VELOCITY (Trip Point)

VELOCITY (Trip Point)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. 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.

13.2.4

13.2.4 TIME Mode (Cooling)

13.2.413.2.4

TIME Mode (Cooling)

TIME Mode (Cooling)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 40msec 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

DELAYDELAY

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

13.2.5

13.2.5 Delay Mode (

13.2.513.2.5 In TIME

Delay Mode (Cooling)

Delay Mode (Delay Mode (

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

TIMETIME

Cooling)

Cooling)Cooling)

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.”

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

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 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 (40ms 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).

13.2.6

13.2.6 PRESSURE Adjustment

13.2.613.2.6

PRESSURE Adjustment

PRESSURE AdjustmentPRESSURE 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.

13.2.7

13.2.7 Filament Width

13.2.713.2.7

Filament Width

Filament WidthFilament 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.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

13.3

13.3 Patch Pipette Fabrication

Patch Pipette Fabrication

13.313.3

Patch Pipette FabricationPatch Pipette Fabrication

Pipettes used for the electrophysiological recording technique of “patch clamping” are characterized by a short, stubby shank 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-97 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.

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.

13.3.1

13.3.1 HEAT

13.3.113.3.1

HEAT

HEATHEAT

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.

13.3.2

13.3.2 PULL Strength

13.3.213.3.2

PULL Strength

PULL StrengthPULL Strength

There is a constant gravitational pull on the carrier bars that can be felt when loading the glass. This pull is usually adequate to form fairly 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 multiline program (see below).

13.3.3

13.3.3 VELOCITY (Trip Point)

13.3.313.3.3

VELOCITY (Trip Point)

VELOCITY (Trip Point)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 pipette’s tip will be and the shorter it’s taper. However, too many cycles can lead to variability. Generally speaking, it is advisable to keep the number of heating cycles to 5 or less.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

13.3.4

13.3.4 TIME Mode (Cooling)

13.3.413.3.4

TIME Mode (Cooling)

TIME Mode (Cooling)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.

13.3.5

13.3.5 PRESSURE

13.3.513.3.5

PRESSURE

PRESSUREPRESSURE

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.

< PULL > key is pressed the air solenoid is opened

13.4

13.4 Step

Step----by

by----Step Patch Pipette Programming

13.413.4

StepStep

Step Patch Pipette Programming

byby

Step Patch Pipette Programming Step Patch Pipette Programming

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:

Filament Type

Filament Type HEAT

Filament TypeFilament Type

Trough R+15 0 *50 150

Box R 0 *30 250

PRESSURE

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

PRESSUREPRESSURE

****The VELOCITY

VELOCITY value will need to be manipulated.

VELOCITYVELOCITY

HEAT PULL

HEATHEAT

PULL VELOCITY

PULLPULL

VELOCITY

VELOCITYVELOCITY

TIME

TIMETIME

2. 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 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.

3. 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

increases, the number of loops dec

increases increases

decreases

decdec

reases.

reasesreases

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

line by

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

number of loops increases

increases.

increasesincreases

decreases, the

decreasesdecreases

5. 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.

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

changes made in a one-line program are amplified throughout the program and can produce 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:

7. 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 values:

HEAT

HEAT PULL

HEATHEAT

PULL VELOCITY

PULLPULL

VELOCITY TIME

VELOCITYVELOCITY

TIME

TIMETIME

Loops 4 times 285 0 45 150

would be written into an equivalent 4 line program:

HEAT

Line 1 285 0 45 150

Line 2 285 0 45 150

Line 3 285 0 45 150

HEAT PULL

HEATHEAT

PULL VELOCITY

PULLPULL

VELOCITY TIME

VELOCITYVELOCITY

TIME

TIMETIME

Line 4 285 0 45 150

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

and the resulting pipette.

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

For larger diameter tips: Decrease HEAT in last line

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

5 to 10 units, or increase/decrease VELOCITY in last line

line by 5 to 10 units, or add a small amount of PULL

lineline (10 to 20) to last line

last line.

last linelast line

last line.

last linelast line

next to last

next to lastnext to last

last

last last

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

13.5

13.5 Technical Tips for Pulling Micropipettes

Technical Tips for Pulling Micropipettes

13.513.5

Technical Tips for Pulling MicropipettesTechnical Tips for Pulling Micropipettes

13.5.1

13.5.1 Regulating the Time it ta

13.5.113.5.1 HEAT.

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

HEAT.HEAT.

Regulating the Time it takes to pull a Sharp Pipette

Regulating the Time it taRegulating the Time it ta

kes to pull a Sharp Pipette

kes to pull a Sharp Pipettekes to pull a Sharp Pipette

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.

13.5.1.1 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”.

13.5.2

13.5.2 Regu

13.5.213.5.2 HEAT.

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

HEAT. HEAT.

Regulating the Length and Tip Size of a Sharp Pipette

lating the Length and Tip Size of a Sharp Pipette

ReguRegu

lating the Length and Tip Size of a Sharp Pipettelating the Length and Tip Size of a Sharp Pipette

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.

13.5.2.1 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.

13.5.2.2 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 Micropip

Micropipette/microinjection needle fabrication

MicropipMicropip

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

ette/microinjection needle fabricationette/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.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

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

cooling may give 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.

13.6

13.6 Fire Polishing

Fire Polishing

13.613.6

Fire Polishing Fire Polishing

The P-97 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 0 0 250

PULL VELOCITY

PULLPULL

VELOCITY TIME

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.

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 set screw protruding from it.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

Figure 13-1. 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 13-2. Fire Polish Spacer in Puller Bar.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

Air Jet

14.

14. HE A TING F IL AMENTS

HEA T IN G FILA ME NTS

14.14 .

HEA T IN G FILA ME NTSHEA T IN G FILA ME NTS

14.1

14.1 General Information

General Information

14.114.1

General InformationGeneral Information

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 your research application, there may be an optimum filament/glass combination that differs from the configuration with which the puller is currently set up. After reading through the following material, if you have questions about which filament to use for your application, contact our technical support staff.

14.2

14.2 Trough Filament

Trough Filament

14.214.2

Trough FilamentTrough Filament

.5mm

2-3mm

Figure 14-1. 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.

14.2.1

14.2.1 Positioning

14.2.114.2.1 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 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 locking screws (A’ and B’ in the glass tubing by turning the appropriate eccentric chrome screw (A or B in See the Filament Replacement section of the Maintenance Chapter for a full description of this adjustment.

Positioning

PositioningPositioning

(A’ and B’ in Figure

(A’ and B’ in (A’ and B’ in

Figure 9999----1111)

FigureFigure

Figure 14

FigureFigure

(A and B in Figure

(A and B in (A and B in

) and then the filament can be moved in relation to

) )

Figure 9999----1111)))). First loosen the two

FigureFigure

(A or B in Figure

(A or B in (A or B in

14----1111)))). This

1414

Figure 9999----1111)))).

FigureFigure

14.2.2

14.2.2 Geometry

14.2.214.2.2 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 14

FigureFigure 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’.

Geometry

GeometryGeometry

14----1111, where the distance between the top corners (distance A) is approximately 2/3 the

1414

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

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 14-1. Trough filament sizes.

Filament

14.3

14.3 Box Filament

Box Filament

14.314.3

Box FilamentBox Filament

Filament Descripti

FilamentFilament

FT316B 1.5mm wide trough

FT320B 2mm wide trough

FT330B 3mm wide trough

FT345B 4.5mm wide trough

Description

DescriptiDescripti

onon

2-3mm

Air Jet

Figure 14-2. 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 trough Note: 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 penetration

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

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 penetration

d aid penetrationd aid penetration

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.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

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.

14.3.1

14.3.1 Positioning

14.3.114.3.1

Positioning

PositioningPositioning

When using a box filament, the glass tubing should be centered vertically and horizontally (Figure

Figure 14

FigureFigure

Figure

Figure 14

FigureFigure

14----2222). This position can be adjusted as described above for the trough filament. See

1414

14----1111 and the Filament Replacement section of the Maintenance section for a full

1414

description of this adjustment.

14.3.2

14.3.2 Geometry

14.3.214.3.2

Geometry

GeometryGeometry

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

FB255BFB255B

or special pulling applications are available upon request.

Table 14-2. 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

Description Glass O.D.

DescriptionDescription

Glass O.D.

Glass O.D.Glass O.D.

FT230B 2mm square x 3.0mm wide 1.0mm

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 ≤ 1.5mm ≤ 2.0mm or 2-3 barrels ≤ 2.0mm or 2-3 barrels ≤ 2.0mm or 2-3 barrels

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

15.

15. MA IN TENAN C E

MAIN TENAN C E

15.15 .

MAIN TENAN C EMAIN TENAN C E

15.1

15.1 Cleaning

Cleaning

15.115.1

CleaningCleaning

To maintain the P-97 in optimal condition the vinyl dust cover that is shipped with the P-97 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 15-1. V-Groove bearings and puller bars.

Occasionally the V-groove bearings (S in slide in their grooves (G in

G in Figure

Figure 15

G in G in

FigureFigure

S in Figure

Figure 15

S in S in

FigureFigure

15----1111) must be cleaned to maintain reproducibility from

1515

15----1111) and the edges of the pull bars that

1515

pull to 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----97!

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

15.2

15.2 Heating Filament Replacement

Heating Filament Replacement

15.215.2

Heating Filament ReplacementHeating Filament Replacement

15.2.1

15.2.1 Systematic Filament Replacement

15.2.115.2.1

Systematic Filament Replacement

Systematic Filament ReplacementSystematic Filament Replacement

97!

97!97!

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

D’ in Figure

Figure 15

D’ in D’ in

FigureFigure

15----2222, only one shown

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

1515

, only one shown, only one shown filament, slip in a new one and center it over the air jet. Then re-tighten the two clamp screws.

15.2.2

15.2.2 Air jet Position

15.2.215.2.2

Air jet Position

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.

15.2.3

15.2.3 Positioning the Filament in Relation to the Glass Capillary

15.2.315.2.3

Positioning the Filament in Relation to the Glass Capillary

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 to achieving the desired pipette’s tip size and shape, and will almost certainly require adjustment after replacing a filament. To make this adjustment:

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.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

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

right carrier bar. Two chrome eccentric screws (A and B in

A and B in Figure

A and B in A and B in

Figure 15

FigureFigure

15----2222) in slots are

1515 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 15-2. Filament alignment.

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

(A’ and B’)

(A’ and B’) (A’ and B’)

6. 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.

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

(A’ and B’)

(A’ and B’)(A’ and B’)

8. 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-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

Line pipettes up at

their shoulders and

lengths.

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 15-3. Micropipette shapes.

15.3

15.3 Pulley Adjustment

Pulley Adjustment

15.315.3

Pulley AdjustmentPulley Adjustment

The position of the two pulleys (F in ((((not shown)

not shown) to the carriers (G in

not shown) not shown)

(F in Figure

Figure 15

(F in (F in

FigureFigure

(G in Figure

Figure 15

(G in (G in

FigureFigure

15----4444A)

A) which carry the cables from the solenoid

1515

A)A)

15----4444A)

A) is adjustable. The pulley position controls

1515

A)A) the 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 15

FigureFigure

15----4444). Taper length inequality is generally caused

1515 by the air 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 15

FigureFigure

15----4444A)

1515

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).

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

Figure 15-4. Pulley Adjustment.

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 15

FigureFigure

15----4444B)

1515

B) before

B) B) the 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 turning the chrome eccentric screws (J in

(J in Figure

Figure 15

(J in (J in

FigureFigure

15----4444A)

A) to move the pulley in small

1515

A)A)

(J’ in Figure

Figure 15

(J’ in (J’ in

FigureFigure

15----4444A)

A) and

1515

A)A)

increments until the two cables are of equal tension. If the carrier no longer stops against its stop in the slot (M in

(M in Figure

Figure 15

(M in (M in

FigureFigure

15----4444A)

A), but stops against the cable, then the cam must be

1515

A)A) adjusted back until the 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.

15.4

15.4 Regeneration of Drierite Granules

Regeneration of Drierite Granules

15.415.4

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-97 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 P-97, 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. The far filter can then also be removed. There is no reason to remove the far aluminum keeper. The Drierite should be spread evenly, one

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

granule deep, on 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 which 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 point, install the air input 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 this point, install the air input At this point, install the air input

puller and turn it on. The air pump will turn on and blow air through the canister. Allow this 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. IIIIt is critical that this dust not be blown into the output tube where it might clog either the air

is critical that this dust not be blown into the output tube where it might clog either the air

is critical that this dust not be blown into the output tube where it might clog either the air is critical that this dust not be blown into the output tube where it might clog either the air solenoid or the air jet.

solenoid or the air jet. After the purging process, you may connect the output tube and

solenoid or the air jet.solenoid or the air jet.

t t

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-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

16.

16. TR OU BLESHO O TING

TROU BL ESHOO T IN G

16.16 .

TROU BL ESHOO T IN GTROU BL ESHOO T IN G

16.1

16.1 Controlling Pipette Tip Shapes

Controlling Pipette Tip Shapes

16.116.1

Controlling Pipette Tip ShapesControlling Pipette Tip Shapes

16.1.1

16.1.1 Problem: What type of glass should be used?

16.1.116.1.1 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.

Problem: What type of glass should be used?

Problem: What type of glass should be used? Problem: What type of glass should be used?

16.1.2

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

16.1.216.1.2

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.

16.1.3

16.1.3 Problem: Okay, but how can tips be made even smaller?

16.1.316.1.3

1. The first thing to try in most cases is to increase the HEAT value. This will generally

2. If the electrode is now too long and results in a resistance too high to pass the necessary

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

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?

Problem: Okay, but how can tips be made even smaller?

Problem: Okay, but how can tips be made even smaller? Problem: Okay, but how can tips be made even smaller?

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.

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

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

in more then 8 seconds, decreasing the cooling may somewhat decrease the tip size. Cooling can be most effectively decreased in the P-97 by decreasing air pressure, however a decreasing TIME may also be useful.

16.1.4

16.1.4 Problem: How can the size of a patch

16.1.416.1.4

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

Problem: How can the size of a patchProblem: How can the size 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

16.1.5

16.1.5 Problem: Patch

16.1.516.1.5

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

Problem: PatchProblem: Patch

pipette tips vary in size from pull to pull.

pipette tips vary in size from pull topipette tips vary in size from pull to

Step----by

by----step patch programming

StepStep

step patch programming” in the PARAMETER

byby

step patch programmingstep patch programming

pull.

pull. pull.

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.

16.1.6

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

16.1.616.1.6

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

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

formed. How can this be done?

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

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.

16.1.7

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

16.1.716.1.7

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?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 bend in the pipette has no effect on the pipette’s tip and should cause no problems unless you are penetrating quite deep in the tissue and you are 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.

16.1.8

16.1.8 Problem: One electrode is much longer than the other electrode.

16.1.816.1.8

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

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

This is caused by one of two things.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

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 Heati

Testing the positionTesting the position

Heating filament replacement

ng filament replacement section of the Maintenance

HeatiHeati

ng filament replacementng 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.

16.1.9

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

16.1.916.1.9

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

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

16.2

16.2 Cont

Controller problems

16.216.2

16.2.1

16.2.1 Problem: The filament does not light up when pull is pressed.

16.2.116.2.1

roller problems

ContCont

roller problemsroller problems

Problem: The filament does not light up when pull is pressed.

Problem: The filament does not light up when pull is pressed.Problem: The filament does not light up when pull is pressed.

There are a number of possible reasons why this might happen.

1. Look and see if the filament has burned out. In some cases, it may be necessary to loosen

the screws holding the filament in place, as a very fine break may be hard to see.

2. If the filament is OK, try running the ramp test and see what happens. If you have just

changed the filament, it is quite possible that the new filament needs a very different HEAT value than what you have been using. It is always a good idea to run the ramp test each time you change the filament.

3. If you can run a ramp test but cannot pull a pipette when using the RAMP value for the

heat setting, there might be an air leak. Check to see if the air compressor is running constantly. If so, try to locate the air leak. Check tubing, connections, air solenoid, and the cap of the drierite canister. If the air compressor is not running, it is possible that the air solenoid is stuck closed or the compressor has failed. Contact Sutter Instruments for further instructions.

4. If you run the ramp test and the HEAT value reaches 999 without the filament heating

up, check the tightness of the exposed connectors in the filament current pathway. These are the filament-holding screws (the screws that hold the two brass jaws and the two nuts connecting the filament wires to the posts behind the filament block).

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

5. If these connectors are tight then the problem is probably the power FETs on the heat

sink. (Contact Sutter Instruments for further instructions.)

16.2.2

16.2.2 Problem: Display blank, fan not on, or locked up/overlap.

16.2.216.2.2

Problem: Display blank, fan not on, or locked up/overlap.

Problem: Display blank, fan not on, or locked up/overlap.Problem: Display blank, fan not on, or locked up/overlap.

1. Check power cord and wall a.c. outlet.

2. If the unit still does not work after verifying it is properly plugged in, remove the power

cord and check the fuse. If the fuse has blown, a failure in components that are not serviceable by the user has likely occurred. Contact Sutter Instrument Company Technical Support.

3. If the fuse is still good, the unit is properly plugged in and it still does not work, a failure

in components that are not serviceable by the user has likely occurred. Contact Sutter Instrument Company Technical Support.

16.2.3

16.2.3 Problem: Display shows a row of blocks.

16.2.316.2.3

Problem: Display shows a row of blocks.

Problem: Display shows a row of blocks.Problem: Display shows a row of blocks.

1. The microprocessor has failed to properly initialize the display. This problem can occur

when the power has been turned off and then on again too rapidly. Press RESET and the display should show the proper power-up message. Always allow at least 5 seconds before turning the power back on. If the display still shows a row of blocks, a failure in components that are not serviceable by the user has likely occurred. Contact Sutter Instrument Company Technical Support.

16.2.4

16.2.4 Problem: Displayed program values are not correct

16.2.416.2.4

Problem: Displayed program values are not correct

Problem: Displayed program values are not correctProblem: Displayed program values are not correct

1. Make sure that values were not changed by another user. Always write down the

program values and the ramp-test value and keep them in a secure place.

2. If the values entered are not held when the power is turned off, a failure in components

that are not serviceable by the user has likely occurred. Contact Sutter Instrument Company Technical Support.

16.3

16.3 Technical Support

Technical Support

16.316.3

Technical SupportTechnical Support

For further assistance, contact Sutter Instrument Technical Support at:

(415) 883

(415) 883----0128 or info@sutter.com

(415) 883(415) 883

0128 or info@sutter.com

0128 or info@sutter.com0128 or info@sutter.com

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

APP E ND IX A.

APP E ND IX A. F US E REPLA CEMEN T

APP E ND IX A.APPEND I X A.

FUS E REPLA C EMENT

FUS E REPLA C EMENTFUS E REPLA C EMENT

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.

Figure 16-1. Power entry module.

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. It is concealed in a compartment as shown in Figure

Figure 16

FigureFigure

16----2222. To remove the spare fuse, press down

1616 on the end of the compartment and push it out of the other end. The old fuse can serve as a convenient tool for pushing the spare fuse compartment out. 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.

Figure 16-2. Fuse holder.



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:

5 x 20 mm glass tube

5 x 20 mm glass tube 5 x 20 mm glass tube

At 110

At 110----120V, Medium Time Delay

At 110At 110

(Examples: Bussmann GMC

(Examples: Bussmann GMC----3A or GMC

(Examples: Bussmann GMC(Examples: Bussmann GMC

120V, Medium Time Delay or Time Lag, 3A 250V

120V, Medium Time Delay 120V, Medium Time Delay

or Time Lag, 3A 250V

or Time Lag, 3A 250Vor Time Lag, 3A 250V

3A or GMC----3333----R; or Littelfuse 239 003 or 239 003.P)

3A or GMC3A or GMC

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

R; or Littelfuse 239 003 or 239 003.P)

R; or Littelfuse 239 003 or 239 003.P)R; or Littelfuse 239 003 or 239 003.P)

APP E ND IX B.

APP E ND IX B. LI MI TED WA R RA NTY

APP E ND IX B.APPEND IX B.

LIM I TE D WARR A NTY

LIM I TE D WARR A NTYLI MI TE D WARR A 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-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

APP E ND IX C.

APP E ND IX C. DI SCLAIM

APP E ND IX C.APPEN D IX C.

DIS C LA IMER

DIS C LA IMDISC L AI M

ERER

The pipette puller Model P-97 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.

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

APP E ND IX D.

APP E ND IX D. T E CHNICA L SPECIF IC A T

APP E ND IX D.AP P E ND IX D.

TEC H NI CA L SPEC IF IC ATIO N S

TEC H NI CA L SPEC IF IC ATTE CH NI CA L SPEC IF IC AT

ION S

ION SIONS

Dimensions (H x

Dimensions (H x W x D):

Dimensions (H x Dimensions (H x 30cm x 53cm 36cm

Weight:

Weight: 60 lb.

Weight:Weight: 28 kg

Electrical:

Electrical:Electrical:

Mains voltage 115 V, 60 Hz

Maximum power consumption 370 VA

Mains fuse (rear of cabinet) at 115V 5 x 20 mm glass tube, Medium Time Delay or Time Lag 3A 250V (Examples: Bussmann GMC-3A or

Littelfuse 239 003 or 239 003.P)

Internal safety fuses (not operator accessible) red line Type T (slow blow), 0.5A, 250V,

red/yellow line Type T (slow blow), 0.5A, 250V,

blue line Type T (slow blow), 2.0A, 250V,

orange line Type T (slow blow), 6.25A, 250V,

Power cord 10A, 250V, with safety ground plug

W x D): 12” x 21” x 14”

W x D):W x D):

GMC-3-R; or

3AG



P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

APP E ND IX E.

APP E ND IX E. D R IERITE M AT ER IA L SA

APP E ND IX E.APPENDI X E.

IDENTITY: INDICATING DRIERITE DATE PREPARED 1-3-96 DESCRIPTION: 1/16" TO 1/4" BLUE GRANULES

DRI E RI TE MAT ER IA L SAF ETY DAT A SHEET

DRI E RI TE MAT ER IA L SAD R IERITE M AT ER IA L SA

FETY D AT A SHEET

FETY D AT A SHEETFE T Y DATA SH EET

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-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

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-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

IND E X

IND E XIND EX

AAAA

air

compressor .........................................................17

Drierite canister.................................................18

jet.........................................................................17

nozzle..................................................................17

nozzle adjustment..............................................17

pump...................................................................17

solenoid...............................................................17

valve ....................................................................17

valve solenoid.....................................................17

air mode

delay....................................................................33

time .....................................................................33

air nozzle

alignment............................................................51

air pressure.............................................................32

air valve ..................................................................17

air valve solenoid ...................................................17

angle plate ....................................................... 18, 19

aspiration pipettes .................................................26

BBBB

base .........................................................................21

baseplate.................................................................21

box filaments..........................................................48

bumpers..................................................................20

button

reset.....................................................................13

CCCC

cable ........................................................................21

adjustment................................................... 53, 54

cable pulley assembly

bumpers..............................................................20

cable ....................................................................21

panels, L/R..........................................................20

pull bars..............................................................51

puller bars...........................................................20

spring stops ........................................................20

V-bearings ..........................................................51

cable pulley assembly-bearings ............................21

cables................................................................ 53, 54

cables pulley assembly...........................................20

configuration

default.................................................................27

control functions....................................................31

air pressure.........................................................32

ramp test ............................................................31

controls

front panel ..........................................................13

cycle

definition ..................................................... 14, 25

DDDD

date..........................................................................14

default configuration...................................... 27, 35

delay

definition ..................................................... 15, 26

desiccant

regeneration.......................................................54

dimensions..............................................................67

of glass ..................................................................1

disclaimer ...............................................................65

display.............................................................. 13, 14

vacuum fluorescent display ..............................13

Drierite ...................................................................54

regeneration.......................................................54

Drierite canister.....................................................18

drierite material safety data sheet.......................69

FFFF

features

new........................................................................1

filament

adjustment..........................................................19

filaments.................................................................47

box.......................................................................48

geometry...................................................... 47, 49

positioning................................................... 47, 49

reorder numbers......................................... 48, 49

replacement........................................................51

trough .................................................................47

fire polish......................................................... 26, 44

fuse

holder..................................................................61

location ...............................................................61

replacement........................................................61

spare....................................................................61

fuses

fusesfuses

mains

mainsmains

replacement

replacement......................................................3

replacementreplacement

fuses, replacement

fuses, replacementfuses, replacement

internal ...............................................................67

mains

mains............................................................ 62, 67

mainsmains

GGGG

general information.................................................1

glass

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

dimensions............................................................1

glassware

glasswareglassware

alignment............................................................51

loading ................................................................23

precautions

precautions...........................................................5

precautionsprecautions

specifications........................................................7

HHHH

heat

definition ............................................................14

heat value

optimal setting ...................................................23

heating assembly ...................................................18

angle plate ................................................... 18, 19

eccentrics............................................................19

heating filaments................................See filaments

holding type pipettes.............................................26

IIII

ID...............................................................................1

KKKK

keypad.....................................................................13

keys

CLR.............................................................. 13, 28

ENTR..................................................................13

LAST............................................................ 13, 28

NEXT........................................................... 13, 28

numeric...............................................................13

PULL..................................................................13

STOP ..................................................................13

LLLL

line power (mains).................................................11

locked/unlocked......................................................14

MMMM

mains

mains.......................................................................11

mainsmains

fuses

fuses ......................................................... 3, 62, 67

fusesfuses

power consumption ...........................................67

power cord..........................................................68

voltage.................................................................67

memory...................................................................36

microinjection needle fabrication.........................37

microinjection pipettes..........................................26

microperfusion pipettes ........................................26

micropipette fabrication........................................37

OOOO

PPPP

P-87 programs........................................................33

panels, L/R..............................................................20

parameter

adjustment..........................................................37

Patch pipette fabrication ......................................40

patch pipettes.........................................................26

pipette

aspiration............................................................26

holding (type).....................................................26

microperfusion...................................................26

patch ...................................................................26

pipette

microinjection ....................................................26

power

consumption.......................................................67

power cord

mains...................................................................68

power entry module...............................................61

power switch ..........................................................11

pressure ..................................................................14

setting.................................................................14

program

clearing ...............................................................28

default configuration.........................................35

definition ..................................................... 14, 25

display .................................................................28

editing.................................................................29

memory...............................................................36

parameters .........................................................37

selection..............................................................27

viewing................................................................28

program line

definition ............................................................25

pull

definition ............................................................15

pull cycle.................................................................27

pull time..................................................................43

puller bar................................................................24

adjustment..........................................................21

alignment............................................................20

puller bars ..............................................................20

pulleys

adjustment................................................... 53, 54

RRRR

ramp test ................................................................31

reset.........................................................................13

OD.............................................................................1

operation

operation.................................................................35

operationoperation

first time use ......................................................23

precautions

precautions...........................................................5

precautionsprecautions

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

SSSS

safety warnings................................................. 3, 61

solenoid...................................................................17

spring stops ............................................................20

TTTT

VVVV

technical specifications..........................................67

technical support .....................................................1

time .........................................................................14

definition ..................................................... 15, 26

tip shapes................................................................57

tip size.....................................................................43

troubleshooting......................................................57

trough filaments ....................................................47

UUUU

unpacking.................................................................9

V-bearings ..............................................................21

velocity

definition ............................................................15

special condition.................................................26

voltage

mains...................................................................67

WWWW

warranty.................................................................63

weight .....................................................................67

write protection......................................................14

P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.03 – DOM (20091301)

SUTTER INSTRUMENT P-97 Operation Manual

Specifications and Main Features

Frequently Asked Questions

User Manual