Bio-Rad CHEF-DR II System User Manual
CHEF-DR®II
Pulsed Field
Electrophor esis
Systems
Instruction Manual and
Applications Guide
Catalog Numbers
170-3612
through
170-3729
For Technical Service
Call Your Local Bio-Rad Office or
in the U.S. Call 1-800-4BIORAD
(1-800-424-6723)
Warranty
The CHEF-DR II control module, drive module, chamber, variable speed pump, and
accessories are warranted against defects in materials and workmanship for 1 year. If any
defects occur in the instruments or accessories during this warranty period, Bio-Rad
Laboratories will repair or replace the defective parts at its discretion without charge. The
following defects, however, are specifically excluded:
1. Defects caused by improper operation.
2. Repair or modification done by anyone other than Bio-Rad Laboratories or an authorized
agent.
3. Damaged caused by substituting an alternative chamber or pump.
4. Use of fittings or spare parts supplied by anyone other than Bio-Rad Laboratories
5. Damaged caused by accident or misuse.
6. Damaged caused by disaster.
7. Corrosion caused by improper solvent* or sample.
This warranty does not apply to parts listed below:
1. Fuses
2. Tubing
3. Electrodes
For any inquiry or request for repair service, contact Bio-Rad Laboratories. Inform BioRad of the model and serial number of your instrument.
Important: This Bio-Rad instrument is designed and certified to meet IEC1010-1**
safety standards. Certified products are safe to use when operated in accordance with the
instructiuon manual. This instrument should not be modified or altered in any way. Alteration
of this instrument will:
Void the manufacturer’s warranty
Void the IEC1010-1 safety certification
Create a potential safety hazard
Bio-Rad Laboratories, Inc. is not responsible for any injury or damage caused by the use
of this instrument for purposes other than for which it is intended or by modifications of the
instrument not performed by Bio-Rad Laboratories or an authorized agent.
** IEC1010-1 is an internationally accepted electrical safety standard for laboratory instruments.
Copyright 1992 Bio-Rad Laboratories
All Rights Reserved
1st Revision
* The CHEF-DR II chamber is not compatible with chlorinated hydrocarbons (e.g., chloroform), aromatic hydrocarbons (e.g., toluene,
benzene), or acetone. Use of organic solvents voids all warranties.
Table of Contents
Page
Section 1 Introduction..................................................................................................1
1.1 Safety..........................................................................................................................1
1.2 Overview ....................................................................................................................1
1.3 Specifications .............................................................................................................2
1.4 Description of Major Components ............................................................................3
Section 2 Operation......................................................................................................6
2.1 Instrument Set-up.......................................................................................................6
2.2 Electrophoresis Chamber Operation..........................................................................6
2.3 CHEF-DR II Operation..............................................................................................7
Section 3 Sample Preparation...................................................................................11
3.1 Agarose Block..........................................................................................................11
3.2 Liquid Samples.........................................................................................................11
3.3 Preparation of Agarose Embedded Mammalian DNA............................................11
3.4 Preparation of Agarose Embedded Bacterial DNA.................................................12
3.5 Preparation of Agarose Embedded Yeast DNA......................................................14
3.6 Accelerated Sample Preparation..............................................................................15
3.7 Restriction Enzyme Digestion of Plugs...................................................................15
3.8 Hemocytometer Usage.............................................................................................16
3.9 Estimation of Agarose Plug DNA Concentration ...................................................17
Section 4 Gel Electrophoresis....................................................................................19
4.1 Casting the Gel.........................................................................................................19
4.2 Buffer Circulation and Temperature........................................................................20
4.3 Sample Loading........................................................................................................21
4.4 DNA Size Standards ................................................................................................22
4.5 Electrophoresis.........................................................................................................22
4.6 Separations at Room Temperature...........................................................................23
4.7 Removing and Staining the Gel...............................................................................23
Section 5 Applications................................................................................................23
5.1 Strategies for Electrophoretic Separations...............................................................24
5.2 Pulsed Field Conditions by DNA Size ....................................................................25
5.3 Pulsed Field Conditions by Organism.....................................................................25
5.4 Blotting Megabase DNAs........................................................................................26
5.5 Separation of DNA Size Standards..........................................................................28
Section 6 Maintenance ...............................................................................................29
6.1 Electrode Replacement.............................................................................................29
6.2 Fuses.........................................................................................................................30
6.3 Maintenance of the Electrophoresis Cell.................................................................30
Section 7 Troubleshooting Guide..............................................................................30
Section 8 References...................................................................................................32
Section 9 Instruments and Reagents for Pulsed Field Electrophoresis................38
Section 1
General Information
1.1 Safety
The CHEF-DR II system uses high voltage and current and should be operated with care
at all times. The safety interlocks are for your protection and should not be circumvented. To
avoid shock, set up the CHEF-DR II components in a dry area. Immediately wipe up any
spilled buffers or salt solutions.
When pausing or aborting a run, always check that the current display goes to zero or
displays OFF. This can take 2-5 seconds while the power supply discharges. It is then safe to
remove the lid from the chamber.
Warning: There are high voltages and currents within the chamber, which can be harm-
ful. Do not attempt to circumvent these safety interlocks. Always turn off the power to the
chamber before working within the electrophoresis cell.
The Model 1000 Mini Chiller is ground isolated. Although there is virtually no current
flowing through the Tygon®tubing into the chiller, you should avoid assembling or disassembling the tubing while the CHEF-DR II system is operating during a run.
When inserting or removing the external temperature probe, be sure that the control module is turned off. Do not handle the probe with wet hands.
Definition of Symbols
Caution, risk of electric shock
Caution (refer to accompanying documents)
1.2 Overview
Pulsed field electrophoresis is a technique for resolving chromosome-sized DNA
molecules. By alternating the electric field between spatially distinct pairs of electrodes,
megabase (mb) sized molecules are able to reorient and move at different speeds through the
pores in an agarose gel. Overview articles and specific applications are listed in the references in Section 8.
The CHEF-DR II system is based on CHEF
31,215
(Clamped Homogeneous Electric Fields)
technology. CHEF exhibits two unique properties which have made it the most commonly
used PFGE technique. First, CHEF provides highly uniform, or homogeneous, electric fields
within the gel. This is accomplished using an array of 24 electrodes, which are “clamped” or
held to intermediate potentials to eliminate lane distortion. Thus, lanes are straight. Second,
CHEF generates a 120° reorientation (field) angle, due the hexagonal geometry of the electrode array, which is the optimal angle for separating DNA molecules ranging from 100 kb
to 6 mb in size. In addition, the CHEF-DR II system maintains homogeneous electric fields
using patented Dynamic Regulation
216
(DR). With DR, the electrodes not only generate the
electric field, but also sense changes in local buffer conductivity due to buffer breakdown,
change in buffer type, gel thickness, or temperature. Voltage potentials are readjusted immediately to maintain uniform fields, thus insuring high resolution separations during runs and
between runs. These properties make the CHEF-DR II system a cost-effective instrument for
separating everything from Yeast Artificial Chromosomes (YACs) to cosmid inserts.
1
1.3 Specifications
These specifications pertain to the complete CHEF-DR II system. A system including
the Model 1000 Mini Chiller is also available.
CHEF-DR II Specifications
Drive Module
Dimensions 26 (depth) x 43 (width) x 14 (height) cm
Construction Aluminum chassis
Weight 7.8 kg
Maximum voltage 250 VDC
Maximum current 500 mA
Electrode potentials Dynamically regulated (feedback adjustment)
+/- 0.5%
Input voltage range 90-132 VAC/47-63 Hz/0.7 amps (maximum)
198-264 VAC/47-63 Hz/0.35 amps (maximum)
Fuses 0.5 amp Fast Blow for high voltage output (front panel)
1.0 amp Slow-Blow (100/120 V) or
0.5 amp Slow-Blow (220/240 V)
Environmental
Operating 50 °F (10 °C) to 90 °F (32 °C) temperature 30-80%
humidity
Storage 32 °F (0 °C) to 140 °F (60 °C) temperature 10-90%
humidity
Control Module
Dimensions 13.5 (depth) x 31 (width) x 17 (height) cm
Maximum current 500 mA
Allowable voltage gradients 0.6 - 6 V/cm, in 0.1 V/cm increments
Data entry Keyboard
Power supply 250 V maximum, to allow maximum gradient of
6 V/cm,continuously adjustable
Functional
Switching range 0.1 sec to 65,000 sec (all electronic switching)
Maximum program blocks 2, with automatic execution
Maximum run time 999 hours per block
Input voltage range 90-132 VAC/47-63 Hz/1.7 amps (maximum)
198-264 VAC/47-63 Hz/0.85 amps (maximum)
Fuses 2.0 amp Slow-Blow (100/120 V) or
1.0 amp Slow-Blow (220/240 V)
Environmental
Operating 50 °F (10 °C) to 90 °F (32 °C) temperature 30-80%
humidity
Storage 32 °F (0 °C) to 140 °F (60 °C) temperature 10-90%
humidity
Electrophoresis cell
Dimensions 43 (depth) x 44 (width) x 11 (height) cm, horizontal format
Construction Acrylic
Lid Safety interlocked
Weight 8.5 kg
Electrodes 24, platinum (0.01 inch diameter)
Temperature monitoring Via precision temperature probe mounted through lid
(optional)
2
Accessories included
Variable speed pump 120 V, ground isolated. Flow rate 0.75 liter/min, typical
Casting stand 14 cm x 13 cm
Comb 10 well comb and comb holder
Tygon tubing 365 cm
Sample plug mold 10 slot
Yeast DNA Standard
S. cerevisiae
YNN295, 2 plugs
Chromosomal Grade Agarose 5 grams
Pulsed Field Certified Agarose 5 grams
Leveling bubble 1
Fuses 0.5 Amp Fast Blow, 2
Manual 1
Model 1000 Mini Chiller (Optional)
Weight 14 kg
Construction Aluminum
Dimensions 42 cm long x 23 cm wide x 24 cm high
Cooling capacity 75 watts of input power at 14 °C
Operating range 5 °C - 25 °C
Fuses 3.0 Amp Slow-Blow (100/120 V) or
1.5 Amp Slow-Blow (220/240 V)
Note: This equipment has been tested and found to comply with the limits for a Class A
digital device, pursuant to Part 15 of the FCC rules. These limits are designed to provide
reasonable protection against harmful interference when the equipment is operated in a
commercial environment. This equipment generates, uses, and can radiate radio frequency
energy and, if not installed and used in accordance with the instruction manual, may cause
harmful interference to radio communications. Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to
correct the interference at his own expense.
1.4 Description of Major Components
Drive Module
The drive module is a separate electronic device which distributes and maintains the individual voltages for the 24 electrodes in the electrophoresis cell. It contains the electrode drivers
which provide and maintain the clamped homogeneous electric fields in the electrophoresis
cell. This feature, dynamic regulation (US Patent 4,878,008 issued to Bio-Rad Laboratories),
modulates the potentials so that the proper voltages are maintained regardless of gel size, or
fluctuations in buffer conductivity or temperature.
Control Module
The control module contains the both the power supply function, which provides the voltage for the 24 drivers in the drive module, and the switcher function, which alternates the
electric field in the electrophoresis cell. All run conditions, including initial and final switch
time (ramp), voltage gradient, and run time, are programmed directly into the control module.
In addition, up to 2 consecutively executing blocks of run conditions can be programmed,
increasing the variety of possible separations. The power supply outputs a maximum voltage
gradient of 6 V/cm, or 200 V. The lowest gradient is 0.6 V/cm, or 20 V.
3
4
Figure 1.1 A shows the relative potentials of each electrode pair when the + 60° vector
(indicated by the arrow) is activated. Net field vector is from NW to SE. The highest potentials are found along the SE segment of the hexagon. The potentials gradually decline along
the adjacent segments. The NW segment, directly opposite the SE, has 0 potential, represented in the diagram as negative terminals. When the - 60° angle is activated, the pattern of
electric charges is as shown in Figure 1.1 B. Together, the two pulses result in a 120° included field angle. Other angles will result in values for the relative electrode potentials, according to predetermined values.
Fig. 1.1. Voltage Clamping by the CHEF-DR II system. A. Relative electrode potentials when the + 60°
field vector is activated. B. Relative electrode potentials when the - 60° field vector is activated.
Electrophoresis Cell
The CHEF-DR II electrophoresis cell consists of a 43 x 44 cm (17” x 17.5”) acrylic box
with 24 horizontal electrodes arranged in a hexagon. Gels are electrophoresed horizontally,
submerged under recirculated buffer. A 14 x 13 cm (5.5” x 5”) gel is cast on a platform in a
separate casting stand. The platform (along with the gel) is removed from the casting stand,
and placed in the center of the hexagon. The platform is held in place by a frame positioned
on the chamber floor. A combination wide/long format is available as an accessory (catalog
number 170-3704). DNA migration and buffer flow is in the direction of the arrow mounted
on the lid.
The electrodes are individually wired to the 24 pin computer cable, which in turn connects
to the drive module. The individual electrodes are replaceable for easy maintenance (see
Section 6). Electrodes are 0.01” diameter platinum wire. They are each sealed with an O-ring
and silicone sealant to provide double protection against leakage. The electrodes will wear out
more rapidly when switch times below 1 second are used, and/or when 6 V/cm gradients are
employed.
There are two small chambers below the level of the main chamber floor at the front and
rear of the main chamber. These chambers are used for buffer circulation and priming the
pump. Buffer enters the main chamber through 6 holes in the floor near the top. A flow baffle is located just in front of these holes to prevent gel movement. Buffer exits the chamber
at the front through the T fitting. One arm is for draining, the other for circulation. The base
of the chamber has four leveling screws for even gel submersion in buffer.
The lid contains an interlock for safety. The voltage directly passes from the drive module through a short-path in the lid interlock. If the lid is removed, the current flow is broken
A ON
+ + + +
-
-
WELLS
-
-
+
+
+
+
+
+
+
FLOW
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
B ON
+
WELLS
+ + + +
-
-
-
-
+
+
+
+
+
+
+
+
FLOW
and high voltage to the electrophoresis cell is disrupted. The lid also contains a mount at the
upper right for an external temperature probe, which monitors buffer temperature in the chamber, and regulates cooling by the optional Model 1000 Mini Chiller.
Pump and Accessories
The CHEF-DR II system includes a variable speed pump, which provides a suitable flow
rate of buffer through the chamber. Substitution of other pumps could pose a safety hazard and
cause improper flow, and therefore lower resolution. The pump’s power supply is electrically isolated within the drive module for safety. Its voltage requirement is independent of the line
voltage supplied to the drive module (e.g. 120, 100, 220, or 240 volts). This pump should not
be plugged into any equipment other than the CHEF-DR II drive module.
The pump is connected to Tygon or plastic tubing. This tubing circulates buffer in and out
of the chamber. The tubing may also pass through a water chiller. In this case, the pump
should be located after the chiller, so that buffer flows through the chiller and then to the
pump. Typically, the dial is set at 70, for about 0.75 l/min.
The complete CHEF-DR II system is shown in Figure 1.2.
5
Fig. 1.2. The complete CHEF-DR II system, with electrophoresis cell, control module, drive module,
variable speed pump, and optional Model 1000 Mini Chiller.
Model 1000 Mini Chiller
The Model 1000 Mini Chiller is a stand alone, portable refrigerated apparatus specifically designed for use with the CHEF-DR II system. Electrophoresis buffer is circulated by
the variable speed pump directly through the unique heat exchanger, which is a tube within
a tube. Buffer circulates through the inner stainless steel tube, while liquid refrigerant circulates through the outer copper tube, resulting in rapid and efficient cooling at a rate of
0.75 °C/minute (from ambient temperature to 14 °C). The external temperature probe regulates cooling by the Model 1000 Mini Chiller, resulting in precise maintenance of buffer temperature in the electrophoresis cell.
Section 2
Operation
2.1 Instrument Setup
Place the CHEF-DR II electrophoresis cell on a level surface, with the control module on
top of the drive module and to the right or on a shelf above. The electrophoresis cell should
be positioned with the T connector facing you and the safety interlock to your right. If the
system includes a Model 1000 Mini Chiller, place it to the left of the cell. Place the variable
speed pump at the rear of the cell and connect the plug from the pump to the port labeled
PUMP CONNECTOR in the front of the drive module. Level the electrophoresis cell with the
four leveling feet at each corner of the electrophoresis cell by placing the leveling bubble
(provided) in the center of the base of the cell.
Fig. 2.1. Interconnections between components of the CHEF-DR II system.
Attach the power cords for the control module, drive module, and Model 1000 Mini
Chiller in the back of each instrument. Be sure that both the control module and drive module are powered off. Connect the 25-pin cable from the electrophoresis cell to the port labeled
OUTPUT TO ELECTROPHORESIS CELL on the front panel of the drive module. Connect
the coiled interlock cable from the electrophoresis cell to the jacks labeled TO INTERLOCK
on the drive module.
Electrophoresis
Chamber
CHEF-DR II
Power Module
Variable Speed
Pump
Mini-Chiller
IN
OUT
25 Pin Cable
3/8"Tygon Tubing
Safety Interlock Cable
CHEF-DR II
Control Module
-+-
+
A+B+A+
B+
From Power
Supply
From
Pulsewave
6
Connect the set of black and red cables from the left set of jacks on the control module labeled
POWER SUPPLY (- and +) to the left set of jacks on the drive module labeled FROM POWER
SUPPLY. Connect - to - with the black cable, and + to + with the red cable. Note: the metal
plugs at the ends of these cables have smaller diameters.
Connect the set of red cables from the right set of jacks on the control module labeled
A+ and B+ to the right set of jacks on the drive module labeled FROM PULSEWAVE.
Connect A+ to A+ and B+ to B+.
2.2 Electrophoresis Cell Operation
When using the Model 1000 Mini Chiller, attach approximately 1-2 feet of 1⁄4 inch internal diameter Tygon tubing to both the FLOW IN and FLOW OUT ports on the Model 1000
Mini Chiller, and secure the tubing with plastic clamps (provided). Connect 2 feet of 3⁄8 inch
internal diameter Tygon tubing directly from the left port of the T connector to the 1⁄4 inch
tubing from the FLOW IN of the Model 1000 Mini Chiller using a 3⁄8 to 1⁄4 inch reducer
(provided). The pump should be positioned between the outlet of the Model 1000 Mini Chiller
and the inlet of the electrophoresis cell. Connect the 1⁄4 inch tubing from the FLOW OUT of
the Model 1000 Mini Chiller to the inlet of the CHEF-DR II pump using another 3⁄8 to 1⁄4 inch
reducer. Connect approximately one to two feet of Tygon tubing from the outlet of the pump
to the single connector at the rear of the electrophoresis cell using a 3⁄8 inch connector (provided). When using a general purpose, recirculating water bath as a chiller, attach about 9.5
feet of 3⁄8 inch internal diameter Tygon tubing to the left outlet port of the T connector on the
electrophoresis cell. Connect the other end of the Tygon tubing to the inlet of the CHEF-DR
II pump. Connect two feet of Tygon tubing from the outlet of the pump to the single connector at the rear of the electrophoresis cell. Coil approximately 4-5 feet of the longer tubing
within the bath of the chiller.
Finally, connect a six inch piece of Tygon tubing to the right outlet port of the T connector and clamp the tubing securely closed; this will be used to drain the buffer in the electrophoresis cell.
Insert the gel frame into the positioning holes in the electrophoresis cell. There are two sets
of three positioning holes. Place the frame only in the holes at the bottom corners of the gel,
opposite the wells, so that the frame is centered in the cell. The outermost holes are for the
optional 21 x 14 cm (8.25 x 5 inch) gel format (See casting stand Instruction Manual for more
details).
If the system includes the Model 1000 Mini Chiller, connect the temperature probe to
the port labeled REMOTE SENSOR on the front panel of the Model 1000 Mini Chiller. Insert
the temperature probe into the lid of the electrophoresis cell. Remove the temperature probe
when opening the lid to prevent breakage and for easy access to the electrophoresis cell.
It is desirable to establish the correct buffer flow prior to attempting any electrophoresis
runs. Once the desired flow rate has been achieved, subsequent electrophoresis runs should
be with the same setting on the variable speed pump. Fill the chamber with 2 liters of the
buffer of choice. The desired flow rate of buffer through the electrophoresis cell is approximately 0.75 liter per minute (approximately 70 on the variable speed pump). Turn on the
pump and measure the flow of buffer at the drain port by removing the clamp from the 6”
piece of tubing. Make adjustments to the buffer flow with the variable speed pump.
It is also very beneficial to fine tune the chiller before attempting any electrophoresis
runs. Turn on the chiller and pump approximately 1⁄2 hour before adjusting the temperature.
Initially, it will be necessary to fine tune the temperature setting to achieve a consistent running temperature.
7
2.3 CHEF-DR II Operation
This section describes general operation. See Sections 3 and 4 for sample preparation,
gel casting, gel running, and staining. In the following sections capitalized words refer to
keys, modes, or indicator lights on the CHEF-DR II control module (e.g. RUN TIME) while
boldface words refer to messages on the display (e.g. End).
Power Up
The CHEF-DR II front panel display is shown in Figure 2.2. The front panel consists of
a three segment display, BLOCK, VOLTS/CM, RUN TIME, START/PAUSE, INITIAL
SWITCH TIME, FINAL SWITCH TIME, CURRENT, and RAISE and LOWER keys. On
power up, the unit will beep once and display b-1 with the BLOCK indicator light illuminated, indicating Block 1 is active. The program parameters will be at the default settings.
Fig. 2.2. Front Panel display of the CHEF-DR II system.
Parameter Entry
The CHEF-DR II system has the flexibility of using up to two separate, consecutively
executing Blocks. Each Block has the run parameters of Initial Switch Time, Final Switch
Time, Run Time, and Volts/cm. During a run, Block 1 is run first and then Block 2. To start
entering the run parameters, turn on the control module. The unit will beep once and display
b-1 with the BLOCK indicator light illuminated, indicating Block 1. If not, press LOWER until
b-1 is displayed. Press VOLTS/CM (the VOLTS/CM indicator light will be illuminated), and
enter the desired value with RAISE and LOWER. Press RUN TIME (the RUN TIME indicator light will be illuminated), and enter the desired value with RAISE and LOWER. Press
BLOCK and VOLTS/CM simultaneously (both the BLOCK and VOLTS/CM indicator lights
will be illuminated), and set Initial Switch Time with RAISE and LOWER. Press VOLTS/CM
and RUN TIME simultaneously (both the VOLTS/CM and RUN TIME indicator lights will
be illuminated), and set Final Switch Time with RAISE and LOWER. If more than 1 Block
is desired, press BLOCK and go to Block 2 by pressing RAISE. The unit will display b-2
with the BLOCK indicator light illuminated, indicating Block 2. Continue entering the run
parameters as in Block 1. Below are the limits for each of the run parameters.
8
Block
Adjust from 1-2 Blocks. Block 1 is run first, then Block 2. A run time of 0 will
disable a Block.
Initial Sw. Time
Press BLOCK and VOLTS/CM simultaneously. Adjust from 0.1 second-65,000
seconds (displayed at 65 t).
Final Sw. Time
Press VOLTS/CM and RUN TIME simultaneously. Adjust from 0.1 second-65,000
seconds (displayed at 65 t).
Run Time
Adjust from 0.1 hours-999 hours. A run time of 0 will disable a Block.
Volts/cm
Adjust from 0.6 volts to 6.0 volts in 0.1 volt increments.
Actual Current
Press BLOCK and RUN TIME simultaneously. This will display the current in
milliamps provided by the power supply. This parameter is not adjustable.
Run Program
When the parameters are set, the program may be initiated by pressing
START/PAUSE. Once initiated, the actual current (in mA) will be displayed for 5 seconds,
with the BLOCK, RUN TIME, and START/PAUSE indicator lights illuminated. When the
program is in progress the unit will display the time remaining (hours) in the current Block with
the RUN TIME and START/PAUSE indicator lights illuminated. The current (milliamps)
can be displayed by pressing BLOCK and RUN TIME simultaneously, with the BLOCK,
RUN TIME and START/PAUSE indicator lights illuminated. Once the program is initiated,
it is not possible to edit any of the run parameters. During a run, the RAISE and LOWER
keys are disabled and the remaining keys will operate as follows:
Block
This will display the current Block.
Initial Sw. Time
This will display the set initial switch time for 3 seconds after which the remaining
time for the current Block in progress will be displayed.
Final Sw. Time
This will display the set final switch time for 3 seconds after which the remaining
time for the current Block in progress will be displayed.
Current Switch
This will display the current switch time for 3 seconds after which the
Time remaining time for the current Block in progress will be displayed. This display is activated by pressing BLOCK, VOLTS/CM, and RUN TIME simultaneously.
Run Time
This will display the set run time for the current Block for 3 seconds after which the
remaining time for the current Block in progress will be displayed.
9
Volts/cm
This will display the set voltage gradient for 3 seconds after which the remaining time
for the current Block in progress will be displayed.
Actual Current
This will display the actual current for the Block in progress. The BLOCK and RUN
TIME indicator lights will be illuminated. If the power supply is at current limit (500 mA
maximum), the BLOCK and RUN TIME indicator lights will flash.
Pause/Start Run
Initially, this will initiate the program, and the actual current (in mA) will be displayed
with the BLOCK, RUN TIME, and START/PAUSE indicator lights illuminated. After 5 seconds, the remaining run time (hours) will be displayed with the RUN TIME and
START/PAUSE inidicator lights illuminated. While the program is running, pressing this
key will put the program into PAUSE and the actual current will be displayed with the BLOCK
and RUN TIME indicator lights illuminated, while the START/PAUSE indicator light will
flash. Pressing this key again will resume the program, and the actual current will be displayed for 5 seconds.
In a muti-block program it is possible to examine the run parameters of any Block that is
not currently being displayed by pausing the CHEF-DR II system. While in PAUSE, the
Raise and Lower keys are active to scroll through the two Blocks. Any parameter in either
Block may be displayed by pressing the appropriate key. Editing of run parameters is not
possible once the program has started.
Pausing the Program
The program in progress can be paused to examine set run parameters by pressing
START/PAUSE while the unit is running. When START/PAUSE is pressed, the actual current will be displayed, with the BLOCK and RUN TIME indicator lights illuminated, while
the START/PAUSE indicator light will flash. Warning: There are high voltages and currents within the chamber, which can be harmful. Wait for the displayed current to reach zero
before removing the lid of the electrophoresis cell. To resume the program, press
START/PAUSE, and actual current will be displayed for 5 seconds, after which the remaining time for the current Block in progress will be displayed.
Clearing the Program
It is possible to completely clear every parameter in Blocks 1 and 2 of a current program
to the default settings. This can be done turning off the control module. Individual parameters
may be reset to defaults by pressing RAISE and LOWER simultaneously for 6 seconds (it will
beep once per second).
Program Termination
The program in progress may be manually terminated by holding down START/PAUSE
for three to four seconds. A program can only be terminated while it is in the RUN mode, it
can not be terminated in the PAUSE mode. When the program is terminated it will beep twice
and the unit will display OFF. Pressing START/PAUSE again will start the program from the
beginning.
When the program terminates under the timer control, the RUN TIME and
START/PAUSE indicator lights will go off, it will sound 2 beeps per second for 10 seconds,
and the unit will display End. The run timers will be reset and all parameters will be retained.
The run parameters may be used again as is, or further modified, and the program may be
started again by pressing START/PAUSE.
10
Section 3
Sample Preparation
3.1 Agarose Blocks
Standard procedures for DNA preparation do not yield intact, high molecular weight
DNA molecules. Large DNA molecules (chromosome-sized) are so fragile that they are
sheared by mechanical forces, such as pipetting, during isolation. To prevent breakage of
large DNA molecules, intact cells embedded in agarose are lysed and deproteinized in situ.
219
The agarose matrix protects the embedded DNA from shear forces and provides an easy way
to manipulate samples. Processed genomic DNA-embedded agarose plugs are loaded directly into sample wells of agarose gels.
The most important and difficult task in preparing cells for embedding in agarose is to
obtain the proper cell concentration. Although optical density is frequently used, it is not reliable. Different strains, plasmid content and growth media all contribute to the actual cell number achieved for a particular optical density. Variation in cell number will cause the amount
of DNA per agarose plug to vary leading to over and/or under loading of the sample. To eliminate the need to generate a growth curve for each individual strain, a hemocytometer provides
the most reproducible method for achieving the proper cell concentration for different types
of cells, bacteria, yeast, and fungi. Detailed instructions for the use of a hemocytometer can
be found in Section 3.7.
Sample inserts are cast in Bio-Rad’s disposable plug mold, catalog number 170-3713.
Each sample mold produces up to fifty 10 x 5 x 1.5 mm agarose plugs. The block thickness
allows rapid and efficient diffusion of enzymes during sample preparation and permits samples to be loaded into wells formed with Bio-Rad’s standard well-forming combs without
excessive trimming.
3.2 Liquid Samples
High molecular weight DNA can be prepared by standard procedures. DNA fragments of
up to several hundred kilobases do not require preparation in agarose blocks, but can be added
to the wells in liquid form. When working with DNA in the range of 50-200 kb, it may be necessary to use pipette tips with large openings. When running only liquid samples, the best
resolution and sharpness of bands can be achieved by using a thin well comb (0.75 mm).
3.3 Preparation of Agarose Embedded Mammalian DNA
The buffers, enzymes, and agarose found in the following procedure are provided in the
CHEF Mammalian Genomic DNA Plug Kit (catalog number 170-3591; see Section 9 for
more information).
1. Prepare a cell suspension in isotonic saline or tissue culture medium without fetal bovine
serum. Count the cells and remove 5 x 107cells for each ml of agarose plugs to be made
and place on ice. See Section 3.7 for hemocytometer usage. The 50 well plug mold makes
5 ml of agarose plugs we recommend making slightly more than 5 ml if all fifty wells
are to be utilized.
2. Prepare a 2% CleanCut™agarose (Bio-Rad) solution in sterile water and melt using a
microwave. Equilibrate the solution to 50 °C in a water bath.
3. Centrifuge the cell suspension at 1,000 x g for 5 minutes at 4 °C. Resuspend the cells in
one-half the final volume of plugs to made using Cell Suspension Buffer (10 mM Tris, pH
7.2, 20 mM NaCl, 50 mM EDTA) and equilibrate the cell suspension to 50 °C.
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