Biocomp GRADIENT MASTER 108 Operating Manual

SOFTWARE REGISTRATION

In order for you to receive software updates at no charge as they become
available, please complete this form and fax or email it back to us. We will then send
you the files as email attachments and you can download them from your PC or Mac
into your Gradient Master (model 107), Piston Gradient Fractionator (model 152) or the
Gradient Station (model 153).

BioComp’s fax number:

506-455-6157

BioComp’s email address:

[e-mail]

Please complete the following:

1. Product model and serial number

2. Tube holder sizes

3. Name of lab head

4.

Company/University

5. Address

6.

Phone / Fax

7. Email address

MODEL 108

GRADIENT MASTER™

ver 5.3

OPERATOR'S MANUAL

© 2011 BioComp Instruments, Inc.

650 Churchill Row, Fredericton

New Brunswick, Canada E3B 1P6

Ph: [phone]; [phone];

Fx: [phone]; Email: [e-mail]

Home Page: <http://www.biocompinstruments.com>

TABLE OF CONTENTS

SECTION 1. INTRODUCTION 4

Warranty and service 4
Maintenance 4
Electrical surge protection 4
Goods damaged in transit 4

SECTION 2. SPECIFICATIONS 5

SECTION 3. PARTS DIAGRAMS 6

SECTION 4. ORDERING INFORMATION 7

A. The Gradient Master Gradient Forming System 7
B. The Western Roller Blot Development System 8
C. Magnabase Blotting Bottles 8

SECTION 5. INSTRUMENT SET-UP 9

SECTION 6. PROGRAMMING INSTRUCTIONS 10

A. Levelling the plate 10
B. Recalling a gradient from memory and running it 10
C. Preparing and capping the gradient tubes 11
D. Forming the gradients 12
E. Repeated use of the same gradient 12
F. Developing your own gradient run parameters 13
G. Programming the device. 14
H. Entering gradient run parameters acquired elsewhere 17
I. Fixed angle and vertical rotors:Using the adjustable marker

block and quick-seal tubes. 17

SECTION 7. INTERNET PROGRAMMING INSTRUCTIONS 18

Entering new system software or run parameters 18

SECTION 8. NOTES AND HELPFUL HINTS 21

A. General 21
B. Gradients 21

SECTION 9. TROUBLESHOOTING 21

GRADIENT RUN PARAMETERS LAST PAGES

2

NOTES and FREQUENTLY USED GRADIENTS

3

Rev. March 10, 2011

OPERATOR'S MANUAL

BIOCOMP Model 108 GRADIENT MASTER

Software Version 5.25

SECTION 1. INTRODUCTION

WARRANTY AND SERVICE

BioComp has a policy of complete customer satisfaction. If, during the first thirty (30) days, you are
unhappy with the GRADIENT MASTER, you may return it for a full refund.

BioComp warrants this instrument to be free of defects in workmanship for a period of one year
from the date of receipt for all electronic and mechanical parts. Liability is limited to repairs or
replacement of the unit at BioComp's discretion. This warranty is in lieu of all other warranties either
express or implied.

Claims against this warranty must be made by first contacting BioComp (phone [phone]). At
this time the remedy will be determined. Units returned to BioComp without our knowledge and
permission will not be accepted.

Claims for shortages or damage in transit must be reported to BioComp within ten (10) working days
of the date of receipt. Such claims made after this period cannot be honored.

MAINTENANCE

Other than the tightening of loose screws and the removal of spilled gradient solutions, no user
service is required . The Model 108 contains a computer which should not be tampered with. In the
unlikely event of computer failure, contact BioComp and we will ship you a new printed circuit board
and simple instructions for its installation.

ELECTRICAL SURGE PROTECTION

As with any computer, voltage spikes and power surges can severely damage the sensitive chips in
this instrument. You are strongly encouraged to purchase a surge protector from your local computer
or hardware store and plug the transformer into it. Since surges and spikes can occur at any time, it is
also wise to turn the unit off when not in use to reduce your instrument's exposure to them.

GOODS DAMAGED IN TRANSIT

f the instrument or any of the accessories are damaged when you receive them, it is critical that you
save the shipping carton(s) and contact us immediately. We will inform you of the return procedure
and shipping addresses to put on the box. If the box is damaged to such an extent that returning the
instrument in it would risk further damage, save the box for inspection by the shipper who will be
notified to come and inspect it. Failure to adhere to these instructions will void any insurance claims

we might seek and result in BioComp absorbing unnecessary expenses.

4

SECTION 2. SPECIFICATIONS

Timer:............................................... 0 - 9:59 min:sec, count up or down

0 - 99.9 min, " " " "
0 - 999 min, " " " "

Tilt angle:......................................... 0.0 - 90.0° from vertical

Speed of rotation:........................... 0 - 60 RPM

Memory:......................................... All gradients that have been developed for the rotors the user

purchases are accessible. The user may enter 10 gradients of
their own for any rotor.

Internet programmability:.......... Software upgrades for both the operating system and the

gradient run parameters are independently upgradable by
downloading from a file on a PC.

Dimensions:................................... 17 cm High x 20 cm Deep x 34 cm Wide

Weight:........................................... Base unit 3 Kg; each tube holder - 0.5 Kg

Power requirements:................... 100-240 VAC, 50-60 Hz, 0.75A.

Operating temperatures............. Ambient to 45°C

5

SECTION 3. PARTS DIAGRAMS

6

SECTION 4. ORDERING INFORMATION

A. The Gradient Master™ gradient forming system.

MagnaBase System: Catalog number

Model 108 Gradient Master base unit ................................. 108-1 (100-120V)

108-2 (220-240V)

Includes: 4" MagnaBase rotating steel plate, 2 layering cannula, fine tip marker, 2 hex wrenches, level, SD

card and SD card reader.

Tube Holder and Cap Selection

Order either the MagnaBase (105-9XX) tube holders from the list below by finding the match for your

centrifuge rotor. The 6-place swinging bucket tube holders are supplied with 6 plastic caps for either
rate zonal (suffix -R) or isopycnic (-I) gradients (e.g. 105-925-R for the MagnaBase SW28 holder and a
set of 6 rate zonal caps). The rate zonal caps leave 4 mm above the finished gradient for sample while
the isopycnic caps leave 10 mm. If both long and short caps are needed, use the suffix -IR ((e.g. 105­925-IR). The 8-place vertical tube holders may also be used for swinging bucket tubes with the same
diameter and vice versa. Prices for the vertical tube holders do not include caps, which must be
ordered separately if you plan to use an 8-place holder for both types of rotors (see below).

Tube holders for swinging bucket rotors (6 tube capacity).

Order Tube Size Beckman Dupont/Sorvall Hitachi Kontron
Cat. No mm/in

105-925 25 x 89 SW32 AH-629/38 SRP28SA TST28.38

1 x 3 1/2 SW28 RPS27-2

RPS27

105-916 16 x 102 SW32.1 AH-629/17 SRP28SA1 TST28.17

5/8 x 4 SW28.1 RPS27-3

RPS27

RPS25-3

105-914A 14 x 89 SW41Ti TH-641 TST41.14

9/16 x 3 1/2

105-914B 14 x 95 SW40Ti RPS40T

9/16 x 3 3/4

105-913 13 x 51 SW50.1 AH-650 RPS65T TST55.5

1/2 x 2 SW55Ti RPS55T-2

SW65Ti RPS50-2

RPS40T-2

RPS40
RPS50

105-911A 11 x 60 SW60Ti TST-60.4 RPS56T TST60.4

7/16 x 2 3/8

105-911B 11 x 34 TLS-55 RP55S RP55S

7/16 x 1 3/8

7

Tube holders for Vertical and Near Vertical Rotors (8 tube capacity)

Order Tube Size Beckman Dupont/Sorvall Hitachi Kontron
Cat. No. mm/in

1). 25mm diameter tubes
105-925-8A 25 x 89 VTi 50 RPV50T

1 x 3 1/2 RP50VF

105-925-8B 25 x 60 TV-865B TV-865B

1 x 2 3/8

105-925-8C 25 x 97 TV-850 TV-850

1 x 3 7/8

2). 16mm diameter tubes
105-916-8 16 x 76 VTi 65.1 RP55VF

5/8 x 3 NVT 65 RPV45T

3). 13mm diameter tubes
105-913-8A 13 x 51 VTi 65.2 SRP83VT

1/2 x 2 VTi 65 RP65VT2

VTi 80 RP65VF

NVT 90 RP55VF2

RPV65T

105-913-8B 13 x 66 TV-865 TV-865

1/2 x 2 5/8

105-913-8C 13 x 38 TLN-100

1/2 x 1 1/2

4). 11mm diameter tubes
105-911-8 11 x 32 TLV-100 RP100VT RP100VT

1/2 x 2

Caps and Marker Blocks

To order extra sets of caps or spare marker blocks, use the same catalog number as the tube holder
but change the 4th digit in the catalog number (e.g. 105-X25) to a 4 for rate zonal caps, to a 5 for
isopycnic caps and to a 6 for marker blocks. To order a full set of 6 caps, put the suffix (-6 ) after the
catalog number (e.g. 105-425-6 for a set of 6 rate zonal caps for the SW28).

B. The Western Roller™ blot development system.

Bottles; Choose from the MagnaBase bottle selection below to cover your blotting needs. The possible

combinations of bottles that can be run simultaneously follows.

C. MagnaBase™ blotting bottles.

Maximum Filter Size

Bottle Size Description (Circumference x Height cm) Cat. No.

1.5"D x 1.3"H STRIP RUNNER Polycarb w/ acrylic lid 12 x 2.5 108-12-03

1.5"D x 4.3"H MINIGEL RUNNER Polycarb w/ acrylic lid 12 x 11 108-12-11

1.5"D x 4.3"H AMBIBLOTTER Polycarb w/ acrylic lid 12 x 16 108-12-16

2.7"D x 5.3"H Polycarbonate bottle with clear acrylic lid 21 x 12 108-21-12

2.7"D x 6.3"H Polycarbonate bottle with clear acrylic lid 21 x 16 108-21-16

RADIATION LIDS
a. To order these bottles with the RADIATION LIDS, place the suffix -R after the Catalog Number listed

above; i.e. 108-21-16-R

b. To order the RADIATION LIDS separately, place -RL before the bottle number; i.e. 108-RL-21-16

Combinations (4" Plate);

1) Three 12-03 or 12-11s in any combination.

2) One bottle 21-12 or 21-16.

8

SECTION 5. INSTRUMENT SET-UP

1. After unpacking the instrument, be sure that all the parts listed on the shipping papers are present.
You should find a bag with 3 allen wrenches, 2 cannula, a marker and a round level. There is also an
SD card and SD card reader. The SD card has all the programming files and manuals for backup. It is
not needed to run the Gradient Master

2. This Gradient Master was shipped with a direct drive MagnaBase steel plate that fits directly on the
rotate motor shaft. If it is not installed, follow these instructions to install the plate.

a. Locate the thin allen wrench (3/32") in the Gradient Master accessory kit.

b. Check the drive shaft on the rotate motor. It should have its flat facing the groove in the motor support block. An arrow

on the top of the shaft should be pointing toward the groove. If the shaft is is not properly positioned, follow the
manual instructions to turn the motor on and slowly bring the shaft around.

c. Place the plate onto the rotate motor shaft with the set screw facing the groove (see B. below). It is far enough down when

the set screw lies in the center of the groove. Tighten the set screw, holding the short end in a pair of pliers to increase
the torque applied. To insure that the set screw is exactly perpendicular to the flat, rotate the plate slightly back and
forth as you tighten so the set screw can "find the flat". If the plate ever loosens during use, repeat this procedure.

Flat facing groove

A. Unit as
shipped

B. Direct

drive plate

Rotate

motor shaft

Rotate Motor

Rotate Motor Platform

Groove

Set Screw

3. Locate the plug for the rotate motor wrapped around the tilt platform and plug it into the socket on
the back of the box. Note the red lines on one side of the plug. This side faces AWAY from the white
back. Be sure to match the pins with their holes and push the plug all the way in.

4. Locate the 24 VDC power supply in the box and plug it into the wall with the proper prongs for your
voltage inserted into the slot on the back of the wall unit. Insert the power plug into the socket on the
back of the Gradient Master. Now, turn the power switch on. The display will transiently show
nothing and then

Level the plate
DOWN UP DONE

Place the round bubble level on the plate and use the keypad to center the bubble front to back. Use
the levelling screw on the right edge of the unit to level the unit side to side. When the plate is
completely level, press DONE.

5. Place the MagnaBase tube holder in the CENTER of the steel plate. You are now ready to use the
instrument.

9

SECTION 6. PROGRAMMING INSTRUCTIONS

A. LEVELLING THE PLATE: A CRITICAL FIRST STEP

There are two Levelling functions:

1. When you power up the device (see above)

2. Levelling the plate during use. Press the EXIT key until the display shows the Main Menu;

GRADIENT MASTER108v5.22
GRAD SYST

Press SYST and the press LEVL and repeat the procedure as above. When done, press EXIT to return
to the main menu.

B. RECALLING A GRADIENT FROM MEMORY AND RUNNING IT

NOTE: The Gradient Master is preprogrammed to run your gradients. You do not need to download
any software. The SD card provided is only for backup.

1. When the plate is level, press the DONE key and the display should read:

GRADIENT MASTER108v5.22
GRAD SYST

Press GRAD:

GRADIENT MENU
LAST RCNT LIST NEW EXIT

If you are using a new gradient for the first time, you have two options. You can find it in the LIST, or
you can enter it into the memory (NEW). To locate it in the LIST, press the LIST key:

Select Rotor, Use Arrow
<--> SW41 EXIT

The rotors you purchased tube holders for should appear. If more than three rotors are loaded, press
the arrow key to shift the display until your rotor appears.

Press the key under the rotor you want to use:

Short Sucr 02-28% wv
DOWN UP USE DEL EXIT

The top line summarizes the gradient: Short is the short cap, used for rate zonal runs, Sucr is the
solute, 02-28% wv are the top and bottom percentages of the solute with the measurement units.

The EXIT key can be used at any time to stop the current run or move one level up toward the Main
Menu with each stroke.

The LIST of gradients is stored in memory in a fixed order: First all the Short cap gradients will be
displayed, followed by the all the Long cap gradients. For the short cap, all the sucrose gradients are
listed first, followed by glycerol and then the exotic gradients such as Percoll, Nycodenz, NaCl etc.
Within each solute, the gradients are organized first by lower percent, then high percent then by
measurement (see the gradient list at the back of the manual). Thus, all the 5% gradients are listed
before the 12%, which are listed before the 15%, etc. Press and hold the DOWN and UP keys to scroll

10

through the list.

When you have found the gradient you desire, press the USE key

00-50% S1/2 5:00/60.0/25
RUN DOWN UP EXIT

Press the DOWN or UP keys to scroll through the steps in this gradient. Press the RUN key when you
are ready to start the run.

A note on steps: Most gradients require mixing at different times/angles/speeds to produce the
desired shape of the gradient. Each step has its own time/angle/speed and they are run in sequence
automatically when you press the RUN key.

C. PREPARING AND CAPPING THE GRADIENT TUBES

1. Introduction. One of the problems we encountered was the wide variation in the capacity of tubes
from different manufacturers, and even in lots from the same source. To avoid problems with specific
volumes in gradient recipes, we now supply each tube holder with a Marker Block which marks the
tube's half full point regardless of source or lot variations. Since you have the choice of using the short
cap for small sample, velocity (rate zonal) runs or the long cap for the large samples used in isopycnic
runs, we now provide a stepped Marker Block to suit both the 10 mm and nm cap types in the open­top swinging bucket tubes.

Note that the gradient run parameters provided with the short caps apply ONLY to gradients run
with the short cap and vice versa.

Half-full mark for short, 4mm cap
Rate zonal runs

2 Step Marker Block

Half-full mark for long, 10mm cap
Isopycnic runs

Insert tube and make mark with
fine tip marker on the tube at the
appropriate step in the marker block

2. Before filling the tubes, place them bottom down into the appropriate Marker Block and use the fine
tip marker provided to make a small "Half-full" mark on the tube where it meets the top of the

block. Use the upper step for the nm cap and the lower step for the 10 mm cap as illustrated above.
This will be your filling guide during layering of the heavy and light solutions.

3. Attach the two cannula provided to syringes and fill all tubes with filtered, degassed, room
temperature light solution ~nm above the half-full mark. Filtering with Whatman #1 just before use is
usually enough to degas as well. With practice, you will see just how much extra light solution to add
so that only a small amount is displaced into the cap. Accuracy is not crucial , in fact you may find it
easier to simply pour the light solution in, especially with the larger volume tubes. However, you
should be careful to avoid leaving bubbles adhering to the wall of the tube.

4. Fill a syringe with filtered, degassed, room temperature heavy solution and insert the cannula quickly
to the bottom of the tube and begin layering the heavy solution. Holding the tube at eye level,
carefully fill until the heavy-light interface rises precisely to the half-full mark. If the tip of the cannula
is kept just under the light interface as it rises, you can layer faster without disturbing the light
solution. Withdraw the syringe quickly and smoothly and then add or remove light solution so that
there is enough to fill air hole when the cap is inserted.

11

For Quick Seal vertical rotor tubes, use an 18 Ga needle-tipped syringe to top off the light solution
and fill the nipple to keep air bubbles from entering the gradient during the run.

5. For open-top tubes, caps are then applied while tilting the tube slightly and aligning the cap's air
escape hole with the last air to leave the tube. The air hole's position is marked with a raised ridge on
the cap. To avoid leaks during insertion, keep the cap's axis parallel to the tube's axis . If the cap is
inserted at an angle, the seal is ineffective. The "extra bit" of light solution added earlier is displaced
into the central reservoir of the caps, eliminating all free air bubbles from the inside of the tube
(small adhered ones are not a problem). Since free-floating bubbles have a very deleterious effect on
gradient formation, their removal is imperative.

Remove excess solution from the reservoir before starting the run.

For the nipple-topped vertical rotor tubes, caps are not needed. Simply top up the light solution
liquid to fill the nipple.

6. At the end of a run, the long or short cap should be removed with a gentle twisting motion so that any
liquid remaining in the air outlet does not disturb the gradient.

7. MagnaBase holders are placed in the center of the plate.
Caution: with the large 1" tubes, an unbalanced tube arrangement in the holder will cause uneven
rotation due to gear "slop", so balance the holder with blank tubes as you would the centrifuge rotor
itself.

8. Once the tubes are capped, you are ready to use the Gradient Master.

D. FORMING THE GRADIENTS

1. As described in Section B above, recall the run from memory and press RUN

05-50% S1/2 5:00/60.0/25
RUN DOWN UP EXIT

2. The plate will tilt to the angle of the first step, pause for 5 sec to allow the two solutions to settle and
begin rotation. The rest is automatic.

05-50% S1/2 5:00/60.0/25
FAST EXIT

3. The FAST key doubles the tilting speed, but should not be used for gradients. When gradient
formation is complete, the plate will return to 0° and give a beep:

05-50% S2/2 4:58/88.0/10
Gradient Done EXIT

4. Remove the caps with an upward twisting motion and then balance the tubes before you add sample.

E. REPEATED USE OF THE SAME GRADIENT

1. A convenient feature is the ability to rerun a gradient with just two key strokes. These are the LAST
and RCNT functions. After levelling the unit on startup, press DONE:

GRADIENT MASTER108v5.22
GRAD SYST

12

Press GRAD:

GRADIENT MENU
LAST RCNT LIST NEW EXIT

2. If you press LAST, the very last gradient run on the device will appear:

Short Sucr 02-28% wv 2St
RUN SW50 EXIT

Place your tubes in the holder and press RUN. It is that simple.

3. To recall a gradient that has been used previously, but was not the last one run, press EXIT to return to
the main menu and then press RCNT:

Select Rotor, Use Arrow
SW28 28.1 SW41 SW40 EXIT

4. When you press the rotor you desire to use, a list of up to 10 gradients will appear in the order of most
recent use. Each time you use a gradient for a given rotor, it goes on top of the list and the other
gradients shift down one position. Use the DOWN and UP keys to scroll through the list as you did in
LIST. When you find the gradient you desire to run, press USE and RUN to begin making the
gradient.

F. DEVELOPING YOUR OWN GRADIENT RUN PARAMETERS

Although you are supplied with a number of gradient profiles, you may find that the gradient you
desire is not on the list. Here we describe the method we use to develop the parameters to enable you
to do the same. First, it is essential that the heavy solution be dyed so that you know when to stop
mixing and so you can evaluate the gradient's shape either visually or with a flow cell. We have had
success with 1-2 ml 0.4% Trypan Blue/100 ml solution or 1-4 ml 0.34% Coomassie Brilliant Blue R/100
ml. (See Coombs and Watts. 1985 Anal. Biochem. 148; 254). For salt gradients (CsCl, NaCl or K
Acetate), use Bromphenol (not bromophenol) blue. India Ink (carbon black) works well in high solute
concentrations since it does not change color. It does tend to aggregate and settle over time, however.

In developing new run parameters, you should be guided by the values in the gradients stored in
memory or at the back of this manual. There is probably already a gradient there close to the one you
plan to make, and you should start by using its listed angle and speed. Time is then the dependent
variable which you will determine empirically, counting up from 0:00 until the gradient is finished.
Should this strategy fail, minor variations in angle and speed will usually produce the gradient you
seek. Remember: the goal is to cease gradient mixing when the first traces of blue dye in the heavy
solution reach the upper corner of the light solution next to the cap.

G. PROGRAMMING THE DEVICE

GRADIENT MENU
LAST RCNT LIST NEW EXIT

1. Press NEW:

Select Rotor, Use Arrow
<--> 28.1 SW41 SW40 EXIT

13

2. Press the rotor you wish to develop a gradient for (only gradients you have the software key for will
appear):

Short Sucr 02-28% wv
EDIT NEXT DONE EXIT

3. In order for the gradient to be stored in the correct location in the LIST, you will need to describe its
basic properties. Words like Short, Sucr and wv are selected from lists that appear when you press the
EDIT key when that character is blinking. Numbers are entered using the numerical keypad. Here
are the words that will appear when you press SCROL after pressing the EDIT key for that character:

CAP SOLUTE LO-HI% UNITS

Short Sucr 0-99 vw
Long Glyc vv
CsCl ww
Dext
Fico
KBr
Koac
Metr
NaAc
NaCl
Nyco
Opti
Perc
Reno
Usr0

Short Sucr 02-28% wv
ENTR SCROL EXIT

4. When the proper value is showing, press ENTR. Pressing the NEXT key will scroll through the values
on the EDIT screen.

5. Pressing EDIT on % light and heavy will bring up the screen:

Short Sucr 02-28% wv
ENTR Use Num Keypad EXIT

6. If you enter a number incorrectly, keep pressing the number keys until the proper values appear.
Press ENTR when the desired value is in place.

7. When all the descriptors are adjusted, press the DONE key:

Short Sucr 02-28% wv
EDIT NEXT DONE EXIT

8. Entering the Run Parameters for each step.

The Model 108 returns to a feature that was present in model 106 that saves a great deal of time and
effort when entering runs with many steps. If you examine at the run parameters at the back of this
manual, you will find runs with 10, 14 and even 21 steps. A few years ago, we discovered that
excellent gradients with high % bottom solutions (>45% sucrose, for example) could be made using a
technique we call START-STOP.
Here is an example from the SW41 catalog: Short cap 5-50% wv sucrose

14

SW41 SHORT SUCR 5-50% wv 11 Steps
Time/Angle/Speed

0:05/87/30
0:15/87/0
0:05/87/30
0:15/87/0
0:05/87/30
0:15/87/0
0:05/87/30
0:15/87/0
0:05/87/30
0:15/87/0
0:19/80/20

Note that the angle stays the same for all but the last step and that the speed alternates between 30
rpm and 0 rpm for 5 repeats: thus the name START-STOP. To simplify data entry, you will only need
to describe the three steps that are different. Each is stored in a separate memory (M1, M2, M3, etc.)
and then run in the desired order.

For example, M1 = 0:05/87/30, M2 = 0:15/87/0, M3 = 0:19/80/20. After programming each memory,
you will set the order of their appearance in your plan. For the 11 step run illustrated, the order is:

1 - 2 - 1 - 2 - 1 - 2 - 1 - 2 - 1 - 2 - 3

IMPORTANT: the time should be entered as 0:00 for any step that you want to leave indeterminate,
typically the last step which is stopped manually as the blue dye starts to penetrate the last bit of the
light solution. Here, since we are unsure what the mixing time for the 11th “polishing” step will be,
M3 should have time set to 0:00 (M3 = 0:00/80/20).

9. Entering the Memories:

You have finished describing the run, now enter the different memories:

Short Sucr 02-28% wv
EDIT NEXT DONE EXIT

Pressing the DONE key brings up the first Memory

Mem 1 0:05m 87.0° 30rpm
TIME NEXT MEM# DONE EXIT

To change the time, press TIME:

Time: 000 min. 05 sec.
DONE MIN. SEC.

05 is blinking. Press 0-7 to change the time to 7 sec, or MIN 0-0-6 to change minutes to 6 min. then
press DONE to return to the next entry, ANGLE:

Mem 1 0:07m 87.0° 30rpm
ANGL NEXT MEM# DONE EXIT

15

Press ANGL, enter the value, press ENTR and the key becomes SPED. Press SPED, enter the value,

press ENTR and it cycles back to TIME.

Press NEXT to scroll between TIME-ANGL-SPED

Note: The DONE key in the TIME entry window might tempt you to press DONE to access ANGLE or
SPEED, but the DONE key on the main screen signifies that all Memories are entered and you are
ready to enter the sequence of memories.

To enter Memory2, press Mem#:

Mem 2 0:15m 87.0° 00rpm
TIME NEXT MEM# DONE EXIT

Enter the values for Mem2, then press Mem# and enter the values for Mem3. Note that higher
Memories (Mem4-9) will have values that are meaningless unless you change them. If you are using
only the first three memories, ignore the higher ones and focus on Memories 1, 2, and 3.

10.Press DONE to enter the Sequencing phase.
The sequence showing when you open this screen will originate from the last run actually used, so if
you want to start with a similar run and modify it, bring up that run from the LIST, run it and
immediately stop it (this enters it in the LAST memory). Use the NEXT and DELete key to cycle
through and remove unwanted memories and then enter the desired memory order.

S#01of01:-------------1­NEXT DEL RUN EXIT

In the example from above, the display shows this after the 11 steps have been entered:

S#12of11:--12121212123-­NEXT DEL RUN EXIT

When you are satisfied with the order, press RUN.

05-50% S01 0:05/87.0/30
FAST STOP HOLD STEP EXIT

The FAST key appears during tilting and disappears when the angle is reached. STOP pauses a run
and is changed to RUN to restart the run.

HOLD stops the timer, but leaves the motors in their current state. The display changes to:

S /1 0:05m 87.0° 30rpm
EDIT NEXT HOLD EXIT

TIME will be blinking. Press the EDIT key, change the time as above and press DONE. Press NEXT,
the angle starts blinking, press EDIT, enter the three digit angle (2,0,0 = 20.0°), press ENTR and plate
tilts to the new angle. Press NEXT to edit Speed in similar fashion.

Press EXIT to leave HOLD and start timing.

Pressing the STEP key advances to the next STEP in the sequence before the current time expires. If
the current step is counting up from 0:00 m or down from a preset time (0:05), the elapsed time when

16

you press the STEP key is changed in that Memory. If that Memory is being used again in the current
run, the original value will appear in each subsequent step using that memory. Only after EXITing a
run and returning to the Memory window will the altered STEPped value be visible.

When the gradient has finished, the display shows:

05-25% S11 1:15/88.7/22
FAST Gradient Done EXIT

Press EXIT once to repeat the run with optional editing, or three times to leave the NEW menu. The
screen asks if you want to save the gradient:

Short Sucr 05-25% wv 1St
SAVE Valid Grad or EXIT

If you press SAVE, the gradient is stored in a special memory that is not replaced when a new LIST is
loaded. It will appear in its proper place in the LIST, as well as in LAST and RCNT. If EXIT is pressed,
the gradient is not added to the LIST.

H. ENTERING GRADIENT RUN PARAMETERS ACQUIRED ELSEWHERE.

The gradient run lists that follow this manual are an expanded version of the gradients stored in LIST.

The extra gradients not in the memory involve special conditions (buffers, temperatures, other
solutes) that could not be represented in the limited display of the device. If you find one you want to
use or acquire it from any other source like a paper or from BioComp, enter in into the rotor memory
using NEW(see above) and it will appear in the LIST for that rotor, and in RCNT and LAST as well.

Follow the procedures in Section G to enter the new gradient.

I. FIXED ANGLE AND VERTICAL ROTORS:

USING THE ADJUSTABLE MARKER BLOCK AND

QUICK-SEAL TUBES

I. Setting the Marker Block:

Since the nipple-tipped Quick Seal tubes can not accept a cap during mixing, we supply the 8-place
fixed angle and vertical rotor tube holders with an adjustable marker block that can be altered to fit
the size of the sample you will load into the tube. The idea is to have the tube full to the very top
during mixing and then to remove the top of the gradient to make room for the sample. Here are the
steps to adjust the marker block to fit your sample volume:

1. Tare an empty tube.

2. Fill it to the top of the nipple with water and reweigh it.

3. Subtract your sample volume (1 ml = 1 g) from the total water weight and divide the remainder by
two. This is the half full volume.

4. Fill a new tube with this half full volume.

5. Loosen the set screw (3/32 allen wrench) on the base of the marker block and insert the half-full
tube.

6. While holding the tube so the bottom of the meniscus is just above the rim of the marker block,
slide the plunger up into the base of the marker block until it just touches the bottom of the tube.
Lock it in place by tightening the set screw. Check the adjustment by reinserting the tube and fine
tune the adjustment as necessary.

II. Forming gradients:

1. Make a half-full mark on the tube using the supplied fine tip permanent marker.

2. Add light solution to just above the line.

3. Layer the heavy solution under the light until the interface lies at the half full line.

4. Top up the light solution until the nipple is full. Use a fine needle-tipped syringe for this.

5. Form the gradient.

17

6. Using weight as your guide, remove the sample volume from the top of the tube, sucking a
mixture of air and water so that you stay at the air-water interface.

7. Carefully layer the sample on top of the gradient and seal the tube.

III. Entering run parameters:

If you have ordered an 8-place tube holder, your Gradient Master will have a rotor called VERT in the
LIST menu of rotors. Its list of runs is initially empty, but will have had one run programmed into it at
the factory. There are a few runs developed specifically for the quick-seal tubes at the end of the run
parameter list in the manual for the same size open top tube. For example, the VTi50 vertical rotor
runs are found at the end of the SW28-32 run parameters, since these two rotors use the same
diameter tube. As it turns out, most of the regular open top swinging bucket rotor runs are nearly the
same as the same run developed for the quick seal tubes of the same diameter. Thus, we have
programmed into your Gradient Master the open top tube runs.

1. Option 1: Use the open top runs as they are. Go to the equivalent open top rotor in LIST, find the
gradient you need and run it.

2. Option 2: Using an open top run, but saving it in the VERT rotor list. Find the run in the open top
LIST (i.e. SW28 Long cap 20-50% Nycodenz) and start and quickly stop the run (this enters the run
parameters into the LAST memory buffer). Go back to LIST, press the VERT rotor key and then press
NEW. The previous (LAST) run will appear in the register. Go through the data entry without
changing anything and Start the run. Exit and Save the run and it will appear in the VERT rotor list.
See Section G. above for the programming instructions.

3. Option 3: Manually entering a Vertical run from the Gradient List in the manual. At the bottom
of the open top list of the same diameter tube, you will find the quick-seal tube runs that have been
developed in that tube. Follow the instructions in Section G. to enter these parameters in the VERT list
of runs as in Option 2 here.

SECTION 7. INTERNET PROGRAMMING INSTRUCTIONS

ENTERING NEW SYSTEM SOFTWARE OR RUN PARAMETERS.

1. Software updates are typically sent by email.

2. Locate the SD card that we shipped with your new Gradient Master. It has the original files on it to
reload old software if ever the need arises. For updates and new rotor keys, you will need to load the
new files on it while removing older versions with the same name. Use the SD card reader (Lexar)
provided with your instrument to load files from the USB port on your PC or Mac

Here is a list of the files as they should be labelled on the SD card:

Cgmcodea.s19 This is the main software version (i.e.5.22)
CGRADINA.s19 This is the current run parameter listing for all rotors (i.e. Gradlist8)
SDGMprga.s19 This tells the software where to put the main program on the flash chip (page 4)
SDGRproga.s19 This tells the software where to load the run parameters (page 1)

The keys that unlock the gradient holders you have purchased are listed as follows: (see the rotor
table on pages 7 & 8 for a complete listing of equivalent rotors).

Crr281a.s19 SW28.1
Crsw28a.s19 SW28, SW32
Crsw40a.s19 SW40
Crsw41a.s19 SW41
Crsw50a.s19 SW50, SW55, SW65
Crsw60a.s19 SW60
Crtl55a.s19 TLS-55

18

Crverta.s19 Empty as shipped but used to manually enter desired vertical rotor runs.

The files are sent as zipped files to prevent file corruption. Follow the directions below to unlock and load

these runs on your Gradient Master.

1. Locate the SD card and its USB reader in the GM kit. Insert the SD card reader in the USB port on your
mac or PC and insert the SD card into the card reader label side up and gold fingers forward.

2. Drag and drop the ZIPPED files (.zip)!to the SD card and open them!by double clicking on it (mac), or
right clicking on it (PC). This !will unzip it, revealing the original file. If a folder appears with files
inside it, make sure these files are removed from the folder and placed on the SD card’s highest level.

3. Close the window and remove the SD card from the reader.

4. Insert the card into your Gradient Master's SD card slot on the front panel

5. Turn the power OFF and then ON with the upper right most key pressed: hold it down until you see
"OK".

BioComp Flash SDLder 1.5
LOAD MDSP GMLD

Press the GMLD key. The display should show:

GRADIENT MASTER SD v1.5
CODE GRAD ROTR

6a. To load new main software, press the CODE key. The display

Downloading SD HEX file.
Rec loaded = 0 -> 2500

After a brief pause, the display starts counting up from 0 again:

Downloading SD CODE file.
Rec loaded = 0 -> 3400

The display then reads:

Downloading SD CODE file.
Wait,COPING Flash Page 4

After ~40 sec, the display reads:

FLASH Programmed, Reset
Wait,COPING Flash Page 4

The main code is now loaded and you should turn the Master off and back on to see if the program
loaded properly. Center the dial, press DONE and the display will show the current program:

GRADIENT MASTER ver3.14L
GRAD SYST

6b. Loading a new GRAD LIST is done in the same fashion. Turn on the unit holding down

the right-most key, wait until the display reads OK, let go and press the GMLD key:

BioComp Flash SDLder 1.5
LOAD MDSP GMLD

19

The display should show:

GRADIENT MASTER SD v1.5
CODE GRAD ROTR

To load new gradient run parameters, press the GRAD key. The display reads:

Downloading SD HEX file.
Rec loaded = 0 -> 2500

After a brief pause, the display starts counting up from 0 again:

Downloading SD CODE file.
Rec loaded = 0 -> 0400

The display then reads:

Downloading SD CODE file.
Wait,COPING Flash Page 1

After ~40 sec, the display reads:

FLASH Programmed, Reset
Wait,COPING Flash Page 1

6c. Now you’ll need to reload the rotor keys, since loading a new Grad List erases all keys you

had previously loaded. Repeat the startup routine and press ROTR:

GRADIENT MASTER SD v1.5
NEXT Rotor: SW28 LOAD

Use the NEXT key to scroll to the rotor you want to load and press LOAD.

Downloading SD HEX file.
Rec loaded = 0000-1300

The display informs you that the key has successfully been loaded:

Version 1.1
Enabled SW28 ROTOR

Repeat this procedure until all the keys are loaded. When you turn the unit off and on and go to the
GRADIENT MENU and press the LIST key, you will see all the rotors you have loaded.

6.!Turn the GM off and back on and you are ready to go.

20

SECTION 8. NOTES AND HELPFUL HINTS

A. GENERAL

1.

If the computer fails, turn the power off, wait 10 seconds and then on to reset it.

2. Any time the angle changes, there is a delay of 5 sec before rotation commences. This allows the tube's
contents to settle before rotation begins.

3. Incubator and cold room use: This instrument is neither designed nor warranted for cold room use
because of its sensitivity to the damaging effects of condensation. Should you wish to use it there, you
do so at your own risk. For above ambient incubations, the entire device can be placed at a maximum
of 45° C indefinitely. Higher temperatures will eventually damage the electronics.

B. GRADIENTS

1. Buffers. Gradient run parameters supplied with each holder will apply to virtually any buffer used in
the 20-200 mM range. If your buffer is radically different from this, it would be best to check rotation
time at the recommended angle and speed using the blue dye method described on page 16.

2. Temperature. Many users wish to run gradients at temperatures higher or lower than 20°C. Since
viscosity of solutions changes with temperature, there will be some effect on gradient formation at
these non-ambient temperatures. We recommend that you form the gradients at room temperature
and then incubate them at the run temperature used.

3. Shelves or cushions are best applied after gradient formation by removing gradient from the bottom
tube with a cannula and replacing it with an equal volume of high density solution. It may not be
necessary to add a shelf to gradients in large tubes containing very dense heavy solution because the
shelf will already be there.

4. Sample volumes are adjustable by removing solution from the top of the tube after gradient
formation or by purchasing the different caps sold by BioComp. Caps leaving 4 or 10 mm for sample
at the top of the tube are now available.

5. Marker blocks are designed so that the tube will be marked at the half-full level. Should you ever
misplace your blocks, the line can easily be measured by 1) taring a dry tube with cap, 2) filling with
water and recapping, 3) removing all water from the cap reservoir and the outside of the tube,
weighing the tube, 5) removing half the water and 6) marking the position of the meniscus. This mark
can then be transferred to other tubes the same distance down from the top with a paper jig.
Replacement marker blocks are available from BioComp.

6. The gradient run lists that follows this manual are an expanded version of the gradients loaded into the
memory. The extra gradients not in the memory involve special conditions (buffers, temperatures,
other solutes) that could not be represented in the limited display of the device. If you find one that
you would like to use, enter in into the rotor memory using NEW(see above) and it will appear in the
LIST for that rotor, and in RCNT and LAST as well.

SECTION 9. TROUBLESHOOTING

Should your unit fail, these are some test procedures you should follow before calling us to determine

the nature of the problem. Here are some fault conditions:

1. The display does not light up on powering up the unit:
You should contact BioComp.

2. There is excessive play in the tilt or rotate motors. The cause is most likely the loosening of the set

21

screws that hold the motor shafts to the tilting motor platform (tilt motor) or the stainless steel drive
spindle that bears the tube holders or the Roller plate. Locate the proper hex wrench in the accessory
kit and tighten down the set screws. The Tilt motor set screw is on the grey motor base on the side of
the box where the tilt motor shaft protrudes (see page 6). To get clear access to it, with the power off,
rotate the motor shaft until the set screw appears in the groove in the motor base.

3. Please help others by reporting problems to BioComp so that we can improve our products. See the
front page for contact information.

SECTION 10. Rotor run parameters follow.

22