Bio-Rad CHEF Mapper XA System User Manual
CHEF Mapper®XA
Pulsed Field
Electrophoresis System
Instruction Manual and
Application Guide
Catalog Numbers
170-3670 to 170-3673
For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)
Warranty
Bio-Rad Laboratories warrants the CHEF Mapper system power module, chamber, variable speed
pump, and accessories against defects in materials and workmanship for 1 year. If any defects occur in
the instrument or accessories during this warranty period, Bio-Rad Laboratories will repair or replace
the defective parts free. 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, bar code reader, pump, or
temperature probe
4. Use of fittings or spare parts supplied by anyone other than Bio-Rad Laboratories
5. Damage 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 Instrument Service at
1-(800) 424-6723. Inform Bio-Rad of the model and serial number of your instrument.
* The CHEF Mapper 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.
Model
Catalog No.
Date of Delivery
Warranty Period
Serial No.
Invoice No.
Purchase Order No.
Table of Contents
Page
Section 1 Introduction..................................................................................................1
1.1 Overview ....................................................................................................................1
1.2 CHEF Mapper System Specifications .......................................................................1
1.3 Description of Major Components ............................................................................3
1.4 Safety ..........................................................................................................................5
Section 2 Operation ......................................................................................................5
2.1 Set-up..........................................................................................................................5
2.2 Operation ....................................................................................................................7
Section 3 Tutorial on the CHEF Mapper System .....................................................7
3.1 Auto Algorithm ..........................................................................................................8
3.2 180° FIGE...................................................................................................................8
3.3 Two State....................................................................................................................9
3.4 Multi State ..................................................................................................................9
3.5 Storing and Recalling a Program in Memory..........................................................10
Section 4 The CHEF Mapper User Interface Display............................................10
4.1 Definitions ................................................................................................................10
4.2 Front Panel................................................................................................................11
4.3 The FIGE Mode .......................................................................................................14
4.4 The Two State Mode................................................................................................17
4.5 The Multi State Mode ..............................................................................................19
4.6 Clock Read and Delay Start .....................................................................................27
4.7 Storage and Recall of Programs...............................................................................28
4.8 Editing Parameters ...................................................................................................29
Section 5 The Auto Algorithm Mode.......................................................................31
5.1 Operation ..................................................................................................................31
5.2 Applications..............................................................................................................32
Section 6 The Interactive Algorithm .......................................................................33
6.1 Introduction ..............................................................................................................33
6.2 Hardware and Software Requirements ....................................................................34
6.3 Operation ..................................................................................................................34
Section 7 Sample Preparation ...................................................................................38
7.1 Agarose Blocks and Liquid Samples.......................................................................38
7.2 Procedure for S. cerevisiae.......................................................................................38
7.3 Procedure for Mammalian DNA .............................................................................39
Section 8 Gel Electrophoresis....................................................................................44
8.1 Casting the Gel .........................................................................................................44
8.2 Buffer Circulation and Temperature........................................................................45
8.3 Sample Loading........................................................................................................46
8.4 DNA Size Standards ................................................................................................46
8.5 Electrophoresis .........................................................................................................46
8.6 Separations at Room Temperature...........................................................................47
8.7 Removing and Staining the Gel ...............................................................................47
Section 9 Applications ................................................................................................47
9.1 Strategies for Electrophoretic Separations...............................................................47
9.2 Ramping Factor ........................................................................................................49
9.3 Multi State Interrupts ...............................................................................................52
9.4 Pulsed Field Conditions by DNA Size ....................................................................52
9.5 Pulsed Field Conditions by Organism .....................................................................53
9.6 Blotting Megabase DNAs ........................................................................................53
Section 10 Maintenance of Equipment.......................................................................56
10.1 Replacing Electrodes................................................................................................56
10.2 Fuses .........................................................................................................................56
Section 11 Troubleshooting Guide ..............................................................................57
Section 12 References ...................................................................................................59
12.1 Applications in Pulsed Field Electrophoresis..........................................................59
12.2 Reference List for Pulsed Field Electrophoresis .....................................................60
Section 13 Product Information..................................................................................68
Copyright, Bio-Rad Laboratories
All Rights Reserved
CHEF technology is licensed to Bio-Rad Laboratories
Safety
The CHEF Mapper 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 Mapper 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 gel box.
The Cooling Module 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 Mapper system is operating during a run.
This instrument intended for laboratory use only.
The CHEF Mapper system conforms to the “Class A” standards for electromagnetic
emissions, intended for laboratory equipment applications. It is possible that emissions from
this product may interfere with some sensitive appliances when placed nearby or on the same
circuit as those appliances. The user should be aware of this potential and take appropriate
measures to avoid interference.
Section 1
Introduction
1.1 Overview
Pulsed field electrophoresis is a powerful technique for resolving chromosomal sized
DNAs.
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Alternating the electric field between spatially distinct pairs of electrodes causes
megabase (mb) size DNAs to re-orient and move at different speeds through the pores in an
agarose gel. The CHEF Mapper system separates large and small DNA fragments with better resolution, speed, and accuracy than traditional pulsed field methods. DNAs ranging from
100 bases to over 10 megabases may be effectively resolved. For example, the chromosomal
DNA of Schizosaccharomyces pombe can be resolved in 1 day using a 106° pulse angle, compared to 2 days at 120°. Everything from Yeast Artificial Chromosomes (YACs) to M13
inserts can be separated with a single instrument. Applications include top down and bottom
up mapping (Not I and cosmid cloning, respectively), electrophoretic karyotyping, analysis of
tumor cell DNA rearrangements, DNA damage and repair, mammalian DNA analysis, separation of linear and circular DNAs, separation of large proteins, and analysis of bacterial,
yeast, and parasite strain homogeneity.
The CHEF Mapper system is based on two leading technologies, CHEF (clamped homogeneous
electric fields)31and PACE (programmed autonomously controlled electrodes).32The system provides highly uniform, or homogeneous, electric fields within the gel, using an array of 24 electrodes,
some of which are clamped, or held to intermediate potentials to eliminate lane distortion. Thus, lanes
are straight. The system maintains uniform fields using patented Dynamic Regulation (US patent number 4,878,008). The electrodes sense changes in buffer conductivity due to buffer breakdown, buffer
type, gel thickness, pH fluctuations, and temperature, and potentials are readjusted immediately to
maintain uniform fields, thus insuring high resolution. In PACE, each electrode’s voltage is controlled
independently by firmware. Whereas other CHEF systems are limited to two vectors and a 120° pulse
angle, the CHEF Mapper system allows up to 15 vectors per block with a total of 8 blocks, each vector with its own voltage, angle, and duration. Thus, the CHEF Mapper system may simulate virtually
any pulsed field technique using homogeneous fields, including FIGE, CHEF, and RFGE. Advanced
programmers may simulate OFAGE and other non-homogeneous field methods using a computer.
The CHEF Mapper system offers innovations beyond original PACE. For example,
nonlinear switch time ramping allows linear separations for many sizes of DNA. Secondary
pulses, or interrupts, unhinge DNAs from obstructions and permit faster separations. The
CHEF Mapper system contains 5 years of protocols embedded on a microchip, eliminating
trial and error in setting parameters. Given the size range you expect to separate, the embedded auto algorithm interrelates the sizes with 10 other variables, and provides the preferred
operating conditions. Common gel and buffer conditions, and run temperature of 14 °C, are
assumed. An Interactive Program Disc provides an extended version of the algorithm. This PC
program allows you to vary gel, buffer, and temperature parameters and print out optimal
conditions. The CHEF Mapper system includes a number of other advanced features, as outlined in the next sections. Overview articles and specific applications are listed in Section 12.
1.2 CHEF Mapper System Specifications
Algorithm
Embedded algorithm for automated optimization of common electrophoresis conditions:
Enter smallest and largest size DNA expected in the sample (range 1 kb to 6 mb). Smallest
fragment is placed approximately 9 cm from the well. Algorithm assumes 1% PFC agarose,
0.5x TBE buffer, 14 °C for DNAs less than 2.5 mb. For DNAs over 2.5 mb, 0.8% PFC
agarose, 1.0x TAE, and 14 °C are assumed.
1
Interactive computer algorithm for full optimization of electrophoresis conditions requires
PC 80386 or compatible, with Microsoft Windows®3.1. Buffer type, buffer concentration,
agarose concentration and type, and buffer temperature, can be varied as inputs.
Power Module
Dimensions 34.5 (depth) x 55.9 (width) x 30.5 (height) cm
Construction Aluminum chassis
Weight 16 kg
Power supply 350 V maximum, to allow maximum gradient of 9 V/cm,
continuously adjustable; built in
Maximum current 0.5 amperes
Allowable voltage gradients 0, and 0.6–9 V/cm, in 0.1 V/cm increments
Battery back up All parameters in memory, up to 3 hr of interruption
Delayed start Up to 72 hr
Electrode potentials Dynamically regulated (feedback adjustment) +/- 0.5% F.S.
Program storage 20 average protocols
Display Fluorescent, 2 lines x 40 characters per line, alpha-numeric
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
Switching Functions
Switching range 50 msec to 18 hr
Switch angle variable 0–360 degrees (all electronic switching) in 0.5° increments
Multistate vector switching Up to 15 vectors per pulse cycle, each definable by angle,
voltage, and duration
Switch time ramps Linear, concave, or convex using hyperbolic function
Secondary pulses Defined by voltage, frequency, angle, and duration
Field inversion (FIGE) Available with asymmetric forward, reverse voltages
Maximum program blocks 8, with automatic execution
Maximum run time 999 hours per block
Fuses 3 Amp Slo-Blo; two each for AC line input
0.5 Amp Fast Blo for high voltage output
Electrophoresis Cell
Dimensions 11.4 (h) x 44.2 (w) x 50.3 (d) cm, horizontal format
Construction Cover Vacuum formed polycarbonate
Base Injection molded polycarbonate
Lid Safety interlocked
Weight 10.2 kg
Electrodes 24, platinum (0.02 inch diameter)
Temperature monitoring via precision temperature probe mounted in base of cell
2
Accessories Included
Variable speed oscillating pump 120 V, ground isolated. Flow rate 1 liter/min, typical
Casting stand 14 cm x 13 cm
Comb 15 well comb and comb holder
Tygon tubing 365 cm
Disposable Sample plug mold 50 slot
Yeast DNA standard
Saccharomyces cerevisiae
YNN295
Chromosomal grade agarose 5 g
Pulsed field certified agarose 5 g
Leveling bubble
Manual
Cooling Module
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 5–25 °C
Fuse 100/120 V: 6.3 amp, 250 Slo-blo
220/240 V: 3.1 amp, 250 Slo-blo
Total system weight 41.7 kg
1.3 Description of Major Components
Power Module
The power module contains the electronics for pulsed field electrophoresis, including a
350 V power supply, the switching functions, and drivers for the 24 electrodes. The front
panel contains a two line fluorescent display, buttons, switches, jacks, and a fuse as described
in Section 4. The fused power supply operates with a maximum of 9 V/cm, or 350 V. The
lowest gradient is 0.6 V/cm, or 20 V. The drivers provide clamped homogeneous electric
fields in the chamber and maintain them regardless of the pulse angle selected. This feature,
dynamic regulation, regulates the potentials so that the proper voltages are maintained
regardless of gel size or fluctuations in buffer conductivity or temperature.
Figure 1.1A 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 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 1.1B. Together, the two pulses result in a 120° included pulse
angle. Other angles will result in similar relative electrode potentials. One such example is field
inversion, shown in Figure 1.2A and 1.2B. In this case, the charges are primarily at N and S.
The pulse angles are 180° and 0°. Appropriately scaled voltages are along the sides of the
hexagon to achieve clamping.
3
Fig. 1.1. Voltage clamping by the CHEF Mapper system. A. Relative electrode potentials when the
+ 60° field vector is activated. B. Relative electrode potentials when the - 60° field vector is activated.
Fig. 1.2. Voltage clamping by the CHEF Mapper system In the FIGE mode. A. Relative electrode
potentials when the 0° field vector is activated. B. Relative electrode potentials when the 180° field
vector is activated.
Electrophoresis Chamber
The CHEF Mapper electrophoresis chamber consists of a 44.2 x 50.3 cm (17.4” x 19.8”)
polycarbonate box with 24 horizontal electrodes arranged in a hexagon. (See Figure 1.3.)
Gels are electrophoresed horizontally, submerged under recirculated buffer. A 14 x 13 cm
(5.5” x 5.1”) gel is cast in a separate casting stand, removed, and placed in the center of the
hexagon. It is held in place by a frame, with pegs which are inserted into holes on the chamber floor. A longer and wider format is available as an accessory. DNA migration and buffer
flow are in the direction of the arrow on the lid.
The heavy duty, 0.02” diameter platinum wire electrodes, replaceable for easy maintenance (see Section 10), are individually connected to the 24 pin computer cable, which in
turn connects to the power module. 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 sec. are used and/or when 9 V/cm gradients are employed.
The two small chambers below the main chamber floor at the front and rear of the
main chamber are used for buffer circulation and priming the pump. Buffer enters the
main chamber through six holes in the floor near the rear. A flow baffle just in front of these
holes prevents gel movement. Buffer exits the chamber at the front through the left port.
The right front port is for draining. The base of the chamber has four leveling screws for
even gel submersion in buffer.
A. + 60° B. - 60°
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The lid contains a safety interlock. Low voltage current from the power module passes
through a short path in the lid interlock. If the lid is opened, the current flow is broken, and
circuitry in the power module shuts off the high voltage. The temperature probe is embedded
in the bottom of the chamber.
Warning: There are high voltages and currents within the chamber which can be
harmful. Do not circumvent the safety interlocks. Always turn off the power to the
chamber before working in the gel box.
Fig. 1.3. A. The complete CHEF Mapper XA chiller system, with chamber and power module, and
B. variable speed pump and Cooling Module.
1.4 Safety
The CHEF Mapper 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 Mapper components in a dry area. Immediately wipe up
any spilled buffers or salt solutions.
When pausing or aborting a run, always check that the high voltage warning light on the
CHEF Mapper system has turned off. In some cases this can take 20 seconds, while the power
supply discharges. It is then safe to remove the lid from the chamber.
The Cooling Module is ground isolated. Although there is virtually no current flowing
through the Tygon tubing into the cooler, avoid assembling or disassembling the tubing while
the CHEF Mapper system is operating with a voltage load.
Section 2
Operation
2.1 Setup
Place the CHEF Mapper electrophoresis chamber on a level surface, with the power module to the right or on a shelf above. Position the electrophoresis chamber with the two quick
releases facing you and the safety interlock to the front. Place the cooling module to the left
of the chamber. Place the variable speed pump at the rear of the chamber, and connect the plug
from the pump to the port labeled PUMP CONNECTOR in the front of the power module.
Level the chamber with the leveling feet at each corner using the leveling bubble provided.
5
AB
Fig. 2.1. Interconnections between components of the CHEF Mapper system.
After making sure the power module is off, attach the power cords for the power module
and Cooling Module to the back of each instrument. Connect the 25-pin cable from the
electrophoresis chamber to the port labeled OUTPUT TO ELECTROPHORESIS CELL on
the front panel of the power module. Connect the coiled interlock cable from the
electrophoresis chamber to the jacks labeled TO INTERLOCK on the power module.
To connect the cell to the Cooling Module, attach approximately 1–2 feet of 1¼4 inch
internal diameter Tygon tubing to both the Flow In and Flow Out ports on the Cooling Module,
and secure the tubing with plastic clamps (provided). Connect a quick release connector (provided) to 2 feet of 3¼8 inch internal diameter Tygon tubing. Attach the quick release connector
to the left front port of the cell. Attach the other end of the 3¼8 inch tubing to the 1¼4 inch
tubing from the Flow In of the Cooling Module using a 3¼8 to 1¼4 inch reducer (provided).
The pump should be positioned between the outlet of the Cooling Module and the inlet (rear)
of the electrophoresis cell. Connect the 1¼4 inch tubing from the Flow Out of the Cooling
Module to the inlet of the pump using a 3¼8 to 1¼4 inch reducer. Connect approximately
two feet of 3¼8 inch Tygon tubing to the outlet of the pump using a 3¼8 to 3¼8 straight connector (provided). Connect a quick release connector to the other end of the 3¼8 inch tubing.
Connect the quick release connector to the inlet of the cell.
Connect a quick release connector to a six inch piece of 3¼8 inch Tygon tubing, and
connect it to the right front port of the cell. This tube will be used to drain the buffer in the
electrophoresis cell.
Connect the 9 pin gray temperature probe cable (included) from the back of the
electrophoresis cell to the Remote Sensor port on the back of the Cooling Module.
Establish the correct buffer flow before attempting any electrophoresis runs. After establishing the flow rate, use that setting on the pump for electrophoresis runs. Fill the chamber
with 2.2 liters of buffer. The optimal buffer flow rate through the electrophoresis chamber is
approximately 1 liter per minute (70–100 setting). Turn on the pump, and measure the flow
of buffer at the right front port. Adjust the buffer flow with the pump.
Fine-tune the Cooling Module before attempting electrophoresis runs. Turn on the cooler
and pump approximately 0.5 hour before adjusting the temperature. Initially, it will be
necessary to fine-tune the temperature setting to achieve a consistent running temperature.
6
2.2 Operation
This section describes general operation. See Section 7 and 8 for sample preparation, gel
casting, gel running, and staining.
1. Pour 2.2 liters of buffer into the electrophoresis chamber.
2. Insert the casting frame into the positioning holes in the electrophoresis chamber. There
are two sets of three positioning holes, depending on the gel size. Do not insert gel stops
in the positioning holes at the top of the gel; they will interfere with the electric field and
cause distortions.
3. Place the gel on the frame. The buffer level inside the frame should be approximately
1–2 mm above the surface of the gel.
4. Turn on the variable speed pump. Maintain a flow rate that is strong enough to keep the
gel inside the frame, but not so strong that it causes the gel to float.
5. Close the lid of the electrophoresis chamber.
6. Check the buffer temperature in the electrophoresis chamber by pressing ACTUAL TEMP
on the Cooling Module panel. Make temperature adjustments at the cooler if necessary.
7. Set the run parameters for the electrophoresis run, following the instructions in Section 4.
If using the auto algorithm, see Section 5.
8. Press START RUN to begin electrophoresis. Gas bubbles should begin to form at the
electrodes. At low field strengths, gas bubble formation is very difficult to see. Check
the current display on the running screen. There should be a finite current reading. If the
current is 0, see Section 11, Troubleshooting.
Section 3
CHEF Mapper System Tutorial
The tutorial demonstrates the five methods in which run parameters can be entered into
the CHEF Mapper system. The entire tutorial will take about 20 minutes, and will familiarize
you with how the run parameters are entered for each method. You should have the unit
completely set up, including the gel chamber with the lid secured (see Section 2).
Turn the instrument on with the power switch at the lower left. The AC power lamp will
light. You will see the current ROM version, followed by the message Welcome to the CHEF
Mapper system. Please enter a command. The tutorial will concentrate on the top panel of
keys (Figure 3.1). For more detailed explanations of the methods described here, refer to
Sections 4–6. Words in bold are as they appear on the CHEF Mapper display, and capitalized
words refer to keys on the control panel.
Fig 3.1. Top panel of keys on the CHEF Mapper system.
7
3.1 Auto Algorithm
This example outlines the steps for separating a DNA sample with a size range of
220 kb–2,200 kb (the size range of S. cerevisiae).
1. Press AUTO ALGORITHM.
2. After Molecular Weight: Low, key in 220, press K-BASES, then ENTER.
3. After Molecular Weight: High, key in 2200, press K-BASES, then ENTER.
4. The lower line will display Calibration Factor [ ], enter for NC (= no change). This
factor alters the run time by the factor entered between 0.1 and 9.9. Press ENTER for
default of 1.0. The lower line displays the physical parameters for the run: 0.5x TBE,
14 °C, 1% PFC agarose. Press ENTER.
5. The screen display shows the calculated electrical parameters: 6 V/cm, Run time= 28:59 (hr),
Included angle 120°. Press ENTER or the down cursor key three times to reach the end
of the screen.
6. The electrical parameters continue on the next screen: Int. Sw. Tm = 26.31s, Fin. Sw.
Tm = 3m48.48s, Ramping factor: a = [ Linear]. The first two entries are the calculat-
ed initial and final switch times. Since the times are different, the CHEF Mapper system
has selected a switch-time ramp. Press ENTER or the down cursor key to move the prompt
past the variables.
7. The final display is A program is in memory. Start the run by pressing START RUN.
3.2 180° FIGE
1. Press 180° FIGE. The message You will destroy last program - Go on? is displayed.
Enter 1 for yes. This removes the last program in working memory. It does not erase any
stored programs.
2. After Forward Voltage Gradient = [ ] V/cm, press 9 and ENTER.
3. After Int. Sw. Time =, press 90, then SECONDS, then ENTER.
4. After Fin. Sw. Time =, press ENTER.
5. After Rev. Voltage Gradient = [ ] V/cm, press 6 and ENTER.
6. After Rev. Int. Sw. Time =, press 30, then SECONDS, then ENTER.
7. After Rev. Fin. Sw. Time =, press ENTER. This has set up a ratio of 3:1, forward to
reverse switch times and also differences between forward and reverse voltage gradients
(field strengths).
8. For Total Run Time=, type 24, then press HOURS and ENTER.
9. The final display is A program is in memory. Press START RUN. The unit will begin
switching, which is verified by bubbles emanating from the electrodes.
10. The display shows the status of the parameters during the run, including the run time,
time remaining, current, switch time, forward or reverse field, voltage gradient, ratio,
actual volts, and ramping factor. Press the up or down cursor keys to switch between dis-
plays during the run.
11. To stop the run, press START RUN and PAUSE/CONT simultaneously. Answer the
prompts by pressing 1, then ENTER.
8
3.3 Two State
1. Press TWO STATE. The message You will destroy the last program - Go on? is
displayed. Enter 1 for yes. This removes the last program in working memory. It does
not erase any stored programs.
2. For Gradient [ ] V/cm, press 6 and ENTER. For Run time=[ ], press 24, then HOURS
and ENTER. After Included angle=, press 120, then ENTER.
3. On the next screen, for Int. Sw. Tm =, press 60, then SECONDS and ENTER. For
Fin Sw Tm =, press 110, then SECONDS and ENTER.
4. Ramping factor a = is displayed next. This refers to the ramping constant that allows non-
linear ramps. The default is a linear ramp, so default by pressing ENTER.
5. The display is A program is in memory.
3.4 Multi State
In this example, we will set up two blocks, each with a different voltage and angle, and
store the program for future use.
1. Press MULTI STATE.
2. The message You will destroy the last program - Go on? is displayed. Press 1 and ENTER.
This removes the last program in working memory. It does not erase any stored programs.
3. The next display shows Blk 1, Run time =, Interrupt [0=no, 1=yes]. The cursor will
be on Interrupt (secondary pulses) first. Enter 0 for no. For Run time, press 12, then
HOURS and ENTER.
4. Set the first block of parameters. For the first state, or vector, the screen will show Blk1St01,
for Block 1, State 1. For V/cm enter 6, for Angle enter 53. Note that this angle is mea-
sured from the vertical; positive angles are measured counterclockwise, and negative angles
clockwise. The reference point for angles is measured from the bottom of a vertical line.
5. Continue with initial and final switch times. After In Tm press 30, then SECONDS. After
Fn Tm press ENTER.
6. After (Ramping constant) a=, press ENTER for the default of a linear ramp.
7. The next display is Continue with another state? 0= No, 1 = Yes. Press 1 and ENTER.
8. The next display is Blk1 St02. For V/cm enter 6, for Angle type -53, for a= press ENTER. The direc-
tions of the two state vectors for this block are paired at 53° and -53° for an included angle of 106°.
9. Repeat step 5.
10. At Continue with another state?, enter 0 for no. At Continue with another block?,
enter 1 for yes.
11. The next display is Blk 2, Run time =, Interrupt [0=no, l=yes]. The cursor will be on
Interrupt (secondary pulses) first. Enter 0 for no. For the Run time press 10, then
HOURS and ENTER.
12. You will now provide the second set of vectors. The screen will show Blk2St1. For
V/cm enter 6, for Angle enter 60.
13. Continue with switch times. For In Tm and Fn Tm press 1, then MINUTES and ENTER.
For a= press ENTER for the default of the linear ramp.
14. At Continue with another state?, enter 1 for yes. Fill in the variables as in steps 12 and
13, except, for the Angle of Blk2St2, enter -60.
9
15. At Continue with another state?, enter 0 for no. Then Continue with another block?,
enter 0 for no. The display is now A program is in memory.
3.5 Storing and Recalling a Program in Memory
In this exercise, you will store the previous program in memory. This operation must be
performed before pressing START RUN or beginning any other operations.
1. Press STORE PROGRAM on the panel.
2. The message Enter stored User Program number: is displayed. Press 1, then ENTER
to continue. The program will be stored under program number 1.
3. To recall the program into working memory, press USER PROGRAM on the panel. The
display shows You will destroy last program - go on? Enter 1 for yes. This removes the
last program in working memory. It does not erase any stored programs.
4. After Enter stored program number or Enter to abort, enter the number assigned in step 2.
5. Begin electrophoresis with START RUN. To view the program, press BLOCK before
pressing START RUN. It will respond with Enter Block Number:. Press 1. The first
line of the program will appear. You may go through the program and edit it. Press
ENTER or the cursor keys as many times as needed to get to the end of the program.
Section 4
The CHEF Mapper User Interface Display
4.1 Definitions
Terminology is derived from Clark et. al
.32
State or Field State: Electric field vector, defined by voltage, angle, and duration. The
direction of the vector defines the path taken by DNA within the gel at any moment in response
to the field.
Block: A sequence of repeatable vectors (field states) which is repeated for a specified time.
From 1 to 15 states comprise a block.
Program: A sequence of 1–8 blocks making up a run.
FIGE Mode: Field Inversion gel electrophoresis, in which the two electric field vectors are 180° apart.
Two State Mode: Operating mode consisting of two field vectors, with each vector having
the same voltage and duration, but separated in direction by a definable included angle.
Multi State Mode: Operating mode consisting of up to 8 blocks, each consisting of 1–15
field vectors of definable angle, voltage, and duration.
Included Angle: The angle between the two vectors in the two state mode; measured relative
to the top of the gel.
Multi State Angle: The angle between a vector and the vertical, relative to the bottom of the gel.
Linear Switch Time Ramp: A linear increase or decrease in the duration of a field state
from the beginning to the end of a block. The steepness or slope of this ramp is dependent on
the difference between the initial and final switching times and total run time.
Nonlinear Switch Time Ramp: A switch time ramp that changes in a nonlinear fashion.
Voltage Gradient: Voltage across electrodes on opposite sides of the hexagon, a distance of
33.5 cm.
10
4.2 Front Panel
Indicator Lights
A. C. Power: Illuminates when power to the CHEF Mapper system is turned on.
Run On: Illuminates when START RUN is activated, and goes out when the run is over or
when the program is in pause mode.
High Voltage: Illuminates during a run, and goes out when the run is over or when the program is in pause mode.
IMPORTANT: Never turn the CHEF Mapper power switch off when the high voltage
light is on. Use STOP RUN to clear the run program, then turn the power off.
Run Pause: Illuminates when PAUSE/CONT is pressed to pause a run. When this light is on,
the run on and high voltage lights will go out. (Note: The high voltage light will take 5–25 seconds to go off, to allow the power supply time to power down.)
Key Descriptions
Auto Algorithm sets the system to run in auto algorithm mode (see Section 5).
180° FIGE sets the system to run in field inversion mode (see Section 4.3).
Two State sets the system to accept two state parameters entered from the
control panel (see Section 4.4).
Multi State sets the system to accept multi state parameters entered from the
control panel (see Section 4.5).
11
Clock Read displays the time and date (see Section 4.7).
Delay Start is used for entering delayed starts (see Section 4.7).
Clock Set. Simultaneously press CLOCK READ and DELAY START to
change the time or date (see Section 4.7).
User Program retrieves stored user programs (see Section 4.8).
The following six keys may not be used in the run mode. These are
editing keys and can be used only after a program is entered into
memory (see Section 4.9).
Block displays the parameters of any of the eight possible blocks.
Run Time displays the run time parameters of any of the eight possible
blocks.
State displays the parameters of any of the 15 possible states in any of the
8 possible blocks.
Switch Interval - initial displays the initial switch time for any selected
state.
Switch Interval - Final displays the final switch time for any selected
state.
Voltage Gradient displays the voltage gradient in any selected state.
Angle displays the angle in selected state.
12
USERı
PROGRAM
BLOCK
INITIAL
FINAL
SWITCH INTERVAL
STATE
RUNı
TIME
ANGLE
VOLTAGE
GRADIENT
Cursor Arrow Keys are used to scroll through display screens in the auto
algorithm and editing mode.
Number Key Pad is used to enter numeric values. The +/- key is used to
enter positive or negative angles in the multi state mode.
Lower is used only to decrease numeric values currently displayed in the
editing mode.
Raise is used only to increase numeric values currently displayed in the editing mode.
Kilobases (K-BASES) is used to enter size values in the auto algorithm
mode.
Megabases (M-BASES) is used to enter size values in the auto algorithm
mode.
Hours is used to enter run times, interrupt frequencies, interrupt lengths, initial switch times, and final switch times.
Minutes is used to enter run times, interrupt frequencies, interrupt lengths,
initial switch times, and final switch times.
Seconds is used to enter interrupt frequencies, interrupt lengths, initial switch
times, and final switch times.
Enter is used to enter values into memory, to scroll through the display
screens in the edit mode, and to exit from an active delay start mode.
Clear Entry (CLR ENTRY) clears the current entry when entering or editing numeric values, prior to pressing ENTER.
Clear Memory (CLR MEM) clears stored user programs (see Section 4.8).
13
CURSOR
0
1
2
3
456
789
+/-
.
LOWER
RAISE
K-BASES
M-BASES
HOURS
MINUTES
SECONDS
ENTER
CLR ENTRY
CLR MEM
Store Program (STORE PGM) stores approximately 20 user programs
(see Section 4.8).
Stop Run, activated by pressing START RUN and PAUSE/CONT
simultaneously, stops the run and displays the message “You will halt
the program and clear it”. Enter 0 (for No) to put the run into the pause
mode. Enter 1 (for Yes) to clear the program. (Note: The high voltage
light will take 5–25 seconds to go off, to allow the power supply time to
power down.) Enter 2 to go to the editing mode (see Section 4.9).
Start Run is used to begin the electrophoresis run. When START RUN
is pressed, the high voltage, AC power, and run on indicator lights will
come on. IMPORTANT: Never turn the CHEF Mapper power switch
off when the high voltage light is on. Use STOP RUN first to clear the
program, then turn the power off after the high voltage light goes off.
Pause/Cont may be pressed during a run to pause the run. The run pause
indicator light comes on and the run on light goes off. The high voltage
light takes 5–25 seconds to go off, to allow the power supply time to
power down. To start the run, press PAUSE/CONT again.
Comma is used to escape any program. Pressing COMMA for 6 seconds (six beeps) will display the message “Total Initialization - All
memory will be erased - Go on?” Enter 0 (for No) to put the run into
the pause mode. Enter 1 (for Yes) to clear the program. Entering 1 will
erase every stored user program. Be sure you really want to erase everything before answering yes. (Note: The high voltage light will take 5–25
seconds to go off to allow the power supply time to power down.) Enter
2 (for Edit) to enter the editing mode (see Section 4.9).
4.3 FIGE Mode
Entering Run Parameters into FIGE
In field inversion gel electrophoresis (FIGE), the two electric fields are 180° apart. FIGE
is useful in resolving small DNA fragments, less than 100 kb. The FIGE key sets the CHEF
Mapper system to perform field inversion electrophoresis. The FIGE mode can be programmed in a variety of ways, including conventional FIGE, or with forward and reverse
fields of different voltages and different switch times. It is also possible to program linear or
nonlinear switch time ramps in the FIGE mode (see Section 9.2 for ramping).
To run gels in the FIGE mode, enter voltage gradient, switch time (ramp or no ramp),
and run time at the appropriate prompts. The prompt moves sequentially from one parameter to the next after ENTER is pressed. The parameters must be entered separately for the
forward and reverse directions. When FIGE is pushed, the following screen is displayed:
]
m
14
STORE PGM
START RUN
PAUSE/CONT
STOP RUN
START RUN
PAUSE/CONT
,
Forward Voltage Gradient = [
V/c
Forward Voltage Gradient: Enter the forward voltage gradient with the number key pad.
Press ENTER. The total voltage applied across the electrodes is obtained by multiplying the
gradient by 33.5 (33.5 cm is the distance across the hexagonal electrode array). The allowable
voltage gradient range is 0.6–9.0 V/cm in 0.1 V/cm increments.
Int. Sw. Tm: To enter the initial switch time for the forward direction, enter the numeric
value, press HOURS, MINUTES, or SECONDS, then ENTER. The acceptable switch time
range is 0.05 seconds–18 hours. Enter the switch time as one time unit, e. g., enter 3 minutes
and 30 seconds as 3.5 minutes or 210 seconds.
Note: If a mistake is made while keying in a value, press CLR ENTRY and start again.
If the value has been entered before the mistake is found, use the editing mode to make
a correction (see Section 4.9).
Fin. Sw. Tm: To enter the final switch time for the forward direction, enter the numeric value,
press HOURS, MINUTES, or SECONDS, then ENTER. The acceptable switch time range is
0.05 seconds–18 hours. Enter the values in the same way as the forward initial switch time. If
the final switch time is the same as the initial switch time, press ENTER without re-entering
the time.
After the data for the forward gradient are entered, the following screen will appear for
the reverse gradient:
Reverse Voltage Gradient: Enter the reverse voltage gradient value, and press ENTER. The
reverse voltage gradient has an allowable range of 0.6–9.0 V/cm in 0.1 V/cm increments.
Int. Sw. Tm: To enter the initial switch time for the reverse direction, enter the numeric value,
press HOURS, MINUTES, or SECONDS, and ENTER. The acceptable switch time range is
0.05 seconds–18 hours.
Fin. Sw. Tm: To enter the final switch time for the reverse direction, enter the numeric value,
press HOURS, MINUTES, or SECONDS, and ENTER. The acceptable switch time range is
0.05 seconds–18 hours. If the final switch time is the same as the initial switch time, press
ENTER without reentering the time.
After the data for the reverse gradient are entered, the following screen will appear:
Total Run Time: Enter the total run time for the FIGE run, and press ENTER. The allowable
run time range is 1 minute–999 hours. Enter the run time as one time unit, either hours or
minutes. When entering the run time, first put in the numeric value, press HOURS or MINUTES, then ENTER.
[
]
[
]
[
m
[
]
15
Reverse Voltage Gradient =
Int.Sw.Tm =
Total Run Time =
F.Ramp: a =
]V/c
F. Ramp: a =: The forward ramp display will appear only if there is a difference between initial and final switch times. The value “a” is the ramping factor and determines the
mathematical shape of the ramp. For more information on the ramping factor, see Section
9.2. To run a linear ramp, press ENTER at the a= prompt.
R. Ramp: a =: The reverse ramp display will appear only if there is a difference between the
initial and final switch times. Enter a value for “a”, and press ENTER.
After the values are entered for this screen, the CHEF Mapper system will display the message: A Program is in memory. Please enter another command. The program that was
just entered is now in short term memory (RAM). If the CHEF Mapper system is turned off,
the program will be lost. The following options are available:
Start Run: Press START RUN to begin the run. If power goes off during a run, the pro-
gram is saved and resumes when power is restored. When the run is completed, only the
AC power light will be on, and the screen displays the message Run is Completed. Press
1 to save, or 0 to clear it. If you do not want the program saved, enter 0. If you want the
program saved, enter 1. The program can be rerun until power is turned off. To store the
program, press STORE PGM (see Section 4.8).
Edit the Program: To check the program just entered for errors or to make corrections,
see Section 4.9.
Store Program: To store the program just entered so that it is available for future use, see
Section 4.8.
Delay Start: To enter a time delay before the run is started, see Section 4.7.
Remove Program and Set Up a New Program: To remove the program just entered,
press either AUTO ALGORITHM, FIGE, TWO STATE, or MULTI STATE. The mes-
sage You will destroy last Program - Go On? will appear. Choose 0 for No, 1 for Yes,
or 2 for edit the program. Press 1 then ENTER to delete the program. Entering 0 saves the
program.
Running FIGE
While a FIGE program is being run, the following screens are displayed:
Screen 1:
Run time: The total run time, in hours and minutes.
Remaining: The time remaining to the end of the run (i.e., a countdown clock).
ma: The current in milliamperes.
Sw. time: The present switch time. This will change during a ramp.
Forward or reverse: Present field direction.
V/cm: The value of the voltage gradient.
16
Run Time = hh:mm
Screen 2:
Ratio: The ratio between the forward and reverse switch times.
Volts: The actual voltage of the run.
Ramp a: The ramping factor.
To move between the two screens during a run, use the ⇑ or ⇓ cursor keys.
4.4 The Two State Mode
Entering Run Parameters Into Two State
The two state mode allows the separation and resolution of almost all DNA size ranges.
The term two state refers to two field vectors. The directions of these two vectors are determined by the included angle. This angle is determined relative to the gel, as shown in Figure
4.1. In the two state mode the angle can be continuously varied between 0 and 180°. Linear
or nonlinear switch time ramps are also possible in this mode.
Flg. 4.1. Orientation of the included angle in the two state mode.
TWO STATE prompts the entry of voltage gradient, run time, included angle, and switch
time (ramp or no ramp). When TWO STATE is pushed, the following screen is displayed:
V/cm: Enter the voltage gradient using the number key pad, and press ENTER. The total
voltage applied across the electrodes is obtained by multiplying the gradient by 33.5
[
[
]
_
17
Ratio: # : # ______Volts Ramp a = _______
State 1State 2
➤
➤
Included
angle
Gradient =
]V/cm Run Time =
(33.5 cm is the distance across the hexagonal electrode array). The allowable voltage gradient range is 0.6–9.0 V/cm in 0.1 V/cm increments.
Run Time: Enter the total run time for the two state run, and press ENTER. The allowable run
time range is 1 minute–999 hours. Enter the run time as one time unit, either hours or minutes.
When entering the run time, put in the numeric value, then press HOURS or MINUTES.
Note: If a mistake is made while keying in a value, press CLR ENTRY, and start again.
If the value has been entered before the mistake is found, make the correction using the
editing mode (see Section 4.9).
Included angle: Enter the included angle, and press ENTER. The angle entered must be in
degrees, between 0 and 180. Caution: the efficacy of included angles < 90° has not been
proven.
After the data for the first screen are entered, the following screen will appear:
Int. Sw. Tm: To enter the initial switch time, enter the numeric value, press HOURS,
MINUTES, or SECONDS, then ENTER. The allowable switch time range is 0.05 seconds–18
hours. Enter the switch time as one time unit, e. g., enter 3 minutes and 30 seconds as 3.5
minutes or 210 seconds.
Fin. Sw. Tm: To enter the final switch time, enter the numeric value, press HOURS,
MINUTES, or SECONDS, then ENTER. The allowable switch time range is 0.05 seconds–18
hours. The values are entered as in the initial switch time. If the final switch time is the same
as the initial switch time, press ENTER.
Ramping factor: a =: The ramping factor display will appear only if there is a difference
between initial and final switch times. The value “a” is the ramping factor and determines
the mathematical shape of the ramp. For more information on the ramping factor, see Section
9.2. To run a linear ramp, press ENTER.
After all the values are entered for this screen, the CHEF Mapper system will display:
A Program is in memory. Please enter another command. The program that was just
entered is now in a short term memory (RAM). If the CHEF Mapper system is turned off, the
program will be lost. The following options are available:
Start Run: Press START RUN, and the run will begin. If power goes off during a run,
the program is saved and resumes when power is restored. When the run is completed,
only the AC power light will be on, and the screen will display: Run is Completed.
Press 1 to save, or 0 to clear it. To delete the program, enter 0. To save the program, enter
1. The program can be rerun until the power is turned off. To store the program, press
STORE PGM (see Section 4.8).
Edit the Program: To check the program just entered for errors or to make corrections,
see Section 4.9.
Store Program: To store the program just entered so that it is available for future use, see
Section 4.8.
Delay Start: To enter a time delay before the run is started, see Section 4.7.
[
[
]
]
18
Int.Sw.Tm =
] FinSw.Tm =
Remove Program and set up a new Program: To delete the program just entered, press
either AUTO ALGORITHM, FIGE, TWO STATE, or MULTI STATE. The message
You will destroy last Program - Go On? will appear. Press 1 then ENTER to delete
the program. Enter 0 to save the program.
Running Two State
While a two state program is being run, the following screens are displayed:
Screen 1:
Run time: The total run time is shown in hours or minutes.
Remaining: The time remaining to the end of the run (i.e.: a countdown clock).
ma: The total electrode current in milliamperes.
Sw. tm: The present switch time. This will change during a ramp.
Angle: Present angle (half the included angle).
V/cm: The value of the voltage gradient.
Screen 2:
Included angle: This displays the included angle.
Volts: The actual voltage of the run (33.5 x gradient).
Ramp factor a: The ramping factor.
To move between the two screens during a run, use the ⇑ or ⇓ cursor keys.
4.5 The Multi State Mode
Entering Run Parameters into Multi State
The multi state mode allows flexibility in designing pulsed field regimens. Unlike the
two state mode, where the included angle between the two states determines the direction of
the field vector, multi state allows up to 15 independent vectors to be run in any combination.
Each vector (or state) has its own angle, voltage, and time of application (switch time). The
direction of each state is determined by its angle relative to a vertical line from the top to the
bottom of the gel, with the top being 180° and the bottom 0° (see Figure 4.2). Angles measured
counterclockwise from 0° are positive, and angles measured clockwise from 0° are negative.
s
r
__ma
19
Run Time = hh:mm Remaining = hh:mm
Included Angle = ____ ____volt
Ramping Facto
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