Bio-Rad CHEF-DR III Variable Angle System User Manual

CHEF-DR®III

Pulsed Field

Electrophor esis Systems

Instruction Manual

and Applications Guide

Catalog Numbers

170-3690

through

170-3703

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 III power 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. Damage caused by substituting an alternative chamber or pump.

4. Use of fittings or spare parts supplied by anyone other than Bio-Rad Laboratories

5. Damage caused by accident or misuse.

6. Damage 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.

toluene, benzene), or acetone. Use of organic solvents voids all warranties.

Table of Contents

Page

Section 1 General Information.....................................................................................1

1.1 Safety............................................................................................................................1

1.2 Overview......................................................................................................................1

1.3 Specifications...............................................................................................................2

1.4 Description of Major Components..............................................................................3

Section 2 Operation........................................................................................................5

2.1 Instrument Set-up.........................................................................................................5

2.2 Electrophoresis Chamber Operation ...........................................................................6

2.3 CHEF-DR III Operation..............................................................................................7

Section 3 Sample Preparation.......................................................................................9

3.1 Agarose Block..............................................................................................................9

3.2 Liquid Samples ..........................................................................................................10

3.3 Preparation of Agarose Embedded Mammalian DNA.............................................10

3.4 Preparation of Agarose Embedded Bacterial DNA ..................................................11

3.5 Preparation of Agarose Embedded Yeast DNA........................................................12

3.6 Restriction Enzyme Digestion of Plugs ....................................................................13

3.7 Hemocytometer Usage ..............................................................................................13

3.8 Estimation of Agarose Plug DNA Concentration.....................................................15

Section 4 Gel Electrophoresis.....................................................................................16

4.1 Casting the Gel...........................................................................................................16

4.2 Buffer Circulation and Temperature .........................................................................17

4.3 Loading the Samples..................................................................................................18

4.4 DNA Size Standards..................................................................................................18

4.5 Electrophoresis...........................................................................................................19

4.6 Separations at Room Temperature ............................................................................19

4.7 Removing and Staining the Gel.................................................................................20

Section 5 Applications..................................................................................................20

5.1 Strategies for Electrophoretic Separations ................................................................20

5.2 Pulsed Field Conditions by DNA Size......................................................................22

5.3 Pulsed Field Conditions by Organism.......................................................................22

5.4 Blotting Megabase DNAs..........................................................................................23

5.5 Separation of DNA Size Standards ...........................................................................25

Section 6 Maintenance.................................................................................................27

6.1 Replacing Electrodes .................................................................................................27

6.2 Fuses...........................................................................................................................27

6.3 Maintenance of the Electrophoresis Cell...................................................................27

Section 7 Troubleshooting Guide ...............................................................................28

Section 8 References.....................................................................................................30

Section 9 Systems, Accessories and Reagents for

Pulsed Field Electrophoresis......................................................................38

Section 1 General Information

1.1 Safety

The CHEF-DR III 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 III 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 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 harmful. Do not attempt to circumvent the 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, avoid assembling or disassembling the tubing while the CHEF-DR III system is operating.

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 DNAs. By alternating the electric field between spatially distinct pairs of electrodes, megabase (mb) sized DNAs are able to reorient and move at different speeds through the pores in an agarose gel. Overview and applications articles are listed Section 8.

The CHEF-DR III system separates large and small DNA fragments with better resolution, speed, and accuracy, than initial pulsed field methods. DNAs from 100 bases to over 10 megabases (mb) 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, mammalian DNA analysis, and testing for bacterial, yeast, and parasite strain homogeneity.

The CHEF-DR III system uses two leading technologies, CHEF (Clamped Homogeneous Electric Fields)

215

and PACE (Programmable Autonomously Controlled Electrodes).

216

The system provides highly uniform, or homogeneous, electric fields within the gel, using an array of 24 electrodes, 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 4,878,008). The electrodes sense changes in local buffer conductivity due to buffer breakdown, change in buffer type, gel thickness, or temperature, and poten-

tials are readjusted immediately to maintain uniform fields, thus insuring high resolution. In PACE, the voltage potential of each of the 24 electrodes is regulated independently. Unlike the CHEF-DR II system, which has a fixed reorientation (field) angle of 120° due the hexagonal geometry of the electrode array, the CHEF-DR III system can generate field angles from 90–120°. In addition, the CHEF-DR III system permits up to three consecutively executing blocks of run conditions with battery backed-up RAM and automatic restart after power interruption.

1.3 Specifications

These specifications pertain to the complete CHEF-DR III system. A system including the

Cooling Module is also available.

CHEF-DR III Specifications

Power Module:

Dimensions 43 (depth) x 48 (width) x 17.5 (height) cm

Construction Aluminum chassis Weight 10 kg Power supply 350 V maximum, to allow maximum gradient of

9 V/cm, continuously adjustable; built in

Electrical

Maximum amps 0.5 amperes Allowable voltage gradients 0.6–9 V/cm, in 0.1 V/cm increments Battery back-up All parameters in memory Electrode potentials Dynamically regulated (feedback adjustment)

+/- 0.5%

Data entry Keyboard

Functional

Switching range 0.1 sec to 65K sec Switch angle variable 90–120 degrees (all electronic switching) in 1°

increments Maximum program blocks 3, with automatic execution Maximum run time 999 hours per block Input voltage range 100–120 VAC/50–60 Hz/4 amps

220–240 VAC/50–60 Hz/2 amps

Fuses 0.5 amp Fast Blow for high voltage output

3.15 amp Slow-Blow (100/120 V) or

1.60 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 11.4 x 44.2 x 50.3 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

Accessories included:

Variable speed pump 120 V, ground isolated. Flow rate 1 liter/min, typi-

cal Casting stand 14 cm x 13 cm Comb 10 well comb and comb holder Tygon tubing 365 cm Disposable sample plug mold 50 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 spares Manual 1 Screened cap 1

Cooling Module (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

Total System Weight 41.7 kg

Note: This equipment complies with the limits for a Class A digital device, pursuant to Part 15 of the FCC rules. These limits 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, and the user will be required to correct the interference at his own expense.

1.4 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 drivers provide clamped homogeneous electric fields in the electrophoresis cell, and maintain them regardless of the field angle selected. This dynamic regulation feature modulates the potentials so that the proper voltages are maintained regardless of gel size, or fluctuations in buffer conductivity or temperature. The fused power supply operates with a maximum voltage gradient of 9 V/cm, or 300 V. The lowest gradient is 0.6 V/cm, or 20 V.

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 field angle. Other angles will result in values for the relative electrode potentials, according to predetermined values.

Figure 1.1. Voltage clamping by the CHEF-DR III system. A. Relative electrode potentials when the + 60° field vector is activated. B. Relative electrode potentials when the - 60° field vector is activated.

Electrophoresis Chamber

The CHEF-DR III electrophoresis cell consists of a 44.2 x 50.3cm (17.4" x 19.8") 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, removed, 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. DNA migration and buffer flow is in the direction of the arrow on the lid.

The heavy duty 0.02" diameter platinum wire electrodes, replaceable for easy maintenance (see Section 6), are individually connected to the 24 pin computer cable,which 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 second are used, and/or when 9 V/cm gradients are employed.

The two small chambers below the level of 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 top. A flow baffle just in front of the holes prevents gel movement. Buffer exits the chamber at the front through the two ports. The right is for draining, the left for circulation. The base of the chamber has four leveling screws for even gel submersion in buffer.

The hinged lid contains a safety interlock. The voltage passes directly from the Power Module through a short-path in the lid interlock. If the lid is opened, the current flow is broken and voltage to the gel chamber is disrupted. The cell also includes an internal temperature probe, which monitors buffer temperature in the chamber and regulates cooling by the Cooling Module.

Pump and Accessories

Each 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 power module for safety. Its voltage requirement is independent of the line voltage supplied to the drive module (e.g. 100, 120, 220, or 240 volts). This pump should not be plugged into any equipment other than the CHEF-DR III power 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 placed 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.

+ + + +

A. + 60° B. - 60°

➤

+ + + +

–

➤

Cooling Module

The Cooling Module is a stand alone, portable refrigerated apparatus specifically for use with the CHEF-DR III system. The variable speed pump circulates electrophoresis buffer 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 temperature probe in the cell regulates cooling by the Cooling Module, resulting in precise maintenance of buffer temperature.

The complete CHEF-DR III system is shown in Figure 1.2.

Fig. 1.2. The complete CHEF-DR III chiller system, with chamber, power module, variable speed pump, and Cooling Module.

Section 2 Operation

2.1 Instrument Setup

Place the CHEF-DR III 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 ports facing you and the lid safety interlock to the rear. If the system includes a Cooling Module, place it 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 on the back of the power module. Level the electrophoresis cell with the leveling feet at each corner by placing the casting platform in the center of the cell, then placing the leveling bubble (provided) on the casting platform. Putting the casting platform in the center of the cell will level the gel with respect to the electrophoresis cell.

Fig. 2.1. Interconnections between components of the CHEF-DR III system.

Attach the power cords for the power module and Cooling Module to the back of each instrument. Be sure the power module is off. Connect the 25-pin cable from the electrophoresis chamber to the port labeled OUTPUT TO ELECTROPHORESIS CELL on the front of the power module. The 25-pin cable has an safety interlock on the end which is attached to the power module. Connect the coiled interlock cable from the electrophoresis chamber to the jacks labeled TO INTERLOCK on the power module.

2.2 Electrophoresis Chamber Operation

To connect the cell to the Cooling Module, attach approximately 1–2 feet of 1⁄4 inch ID Tygon tubing to both the Flow In and Flow Out ports on the Cooling Module, and secure the tubing with the plastic clamps. Connect the quick release connector to 2 feet of 3⁄8 inch ID Tygon tubing. Attach the quick release connector to the left front port of the cell. Attach the other end of the 3⁄8 inch tubing to the 1⁄4 inch tubing from the Flow In of the Cooling Module using the 3⁄8 to 1⁄4 inch reducer. Place the pump between the outlet of the Cooling Module and the inlet (rear) of the Electrophoresis Cell. Connect the 1⁄4 inch tubing from the Flow Out of the Cooling Module to the inlet of the pump using a 3⁄8 to 1⁄4 inch reducer. Connect approximate- ly 2 feet of 3⁄8 inch Tygon tubing to the outlet of the pump using the 3⁄8 to 3⁄8 straight connector. Connect a quick release connector to the other end of the 3⁄8 inch tubing. Connect the quick release connector to the inlet of the cell.

Connect a quick release connector to a 6 inch piece of 3⁄8 inch Tygon tubing, and connect it to the right front port of the cell. This tube will drain the buffer in the cell.

Connect the 9 pin gray temperature probe cable from the back of the cell to the Remote Sensor port on the back of the Cooling Module.

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).

Variable Speed Pump

Tygon T ubing

Temperature Probe Cable

OUT

Cooling

Module

Electrophoresis

Chamber

25 Pin Cable

Variable Speed Pump Cable

CHEF DR III

Power Module

Safety Interlock Cable

TO INTERLOCK

Output to

Electrophoresis Cell

If the system includes the Cooling Module, connect the temperature probe cable to the REMOTE SENSOR port on the rear panel of the Cooling Module. Insert the other end of the temperature probe cable into the rear of the electrophoresis chamber.

Establish the correct buffer flow before attempting any electrophoresis runs. The optimal flow rate of buffer through the electrophoresis chamber is approximately 0.8–1 liter per minute (approximately 70 on the pump). When the correct flow rate has been achieved, use that pump setting for all subsequent electrophoresis runs. Fill the chamber with 2.2 liters of buffer. 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 pump.

It is 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.

2.3 CHEF-DR III Operation

This section describes general operation. See Sections 3 and 4 for sample preparation, gel casting, gel running, and staining.

Power Up

The CHEF-DR III front panel display is divided into two sections (see Figure 2.3.). On the left are, BLOCK, INITIAL SWITCH TIME, FINAL SWITCH TIME, RUN TIME, and RAISE and LOWER keys. On the right are VOLTS/CM, INCLUDED ANGLE, ACTUAL CURRENT, PAUSE/START RUN, and RAISE and LOWER keys. At power up, the left display will show 1 with BLOCK lit, indicating Block 1 is active. The right display will show OFF. The program parameters will be at the default setting or those last set before the power was turned off, since the battery back-up RAM stores the last program entered. If a program was in progress or in PAUSE, the run parameters will be retained and the PAUSE mode will be active (flashing PAUSE light).

Fig. 2.2. Front panel display of the CHEF-DR III system.

Parameter Entry

The CHEF-DR III system has the flexibility of using up to three separate, consecutively executing Blocks. Each Block has the run parameters of Initial Switch Time, Final Switch Time, Run Time, Volts/cm, and Included Angle. During a run, Block 1 is run first, then Block 2, then Block 3. To enter run parameters into Block 1, press BLOCK. The left display should show 1, indicating Block 1. If not, press RAISE or LOWER on the left side of the display panel until 1 is shown. Enter the Initial Switch Time, Final Switch Time, and Run Time with the RAISE and LOWER keys on the left side of the display panel. Enter the Volts/cm, and Included Angle with the RAISE and LOWER keys on the right side of the display panel. If more than 1 Block is needed, then press BLOCK and go to Block 2 by pressing RAISE. The left display will show the number 2, indicating Block 2. Continue entering the run parameters as in Block 1. If a third Block is needed, press BLOCK, go to Block 3 by pressing RAISE, then continue entering the run parameters as in Block 1. Below are the limits for each of the run parameters.

Block Program from 1–3 Blocks. Block 1 is run first, then Block 2, then

Block 3. A run time of 0 disables a Block.

Initial Switch Time Adjust from 0.1–65K seconds. Final Switch Time Adjust from 0.1–65K seconds. Run Time Adjust from 0.1–999 hours. A run time of 0 disables a Block. Volts/cm Adjust from 0.6–9.0 volts in 0.1 volt increments. Included Angle Adjust from 90–120° in 1° increments. Actual Current Displays the current, in mA, provided by the power supply. This

parameter is not adjustable.

Run Program

When the parameters are set, start the program by pressing PAUSE/START RUN. When the program is in progress, the left panel display will show the time remaining (hours) in the current Block with RUN TIME lit, and the right panel display will show the actual current (milliamps) with ACTUAL CURRENT and PAUSE/START RUN lit. After the program is started, 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 Displays the current Block. Initial Switch Time Displays the set initial switch time for 3 seconds, then displays the

remaining time for the current Block.

Final Switch Time Displays the set final switch time for 3 seconds, then displays the

remaining time for the current Block.

Current Switch Displays the current switch time for 3 seconds, then displays

Time the remaining time for the current Block. This display is activated

by pressing INITIAL SWITCH TIME and FINAL SWITCH TIME simultaneously.

Run Time Displays the set run time for the current Block for 3 seconds then

displays the remaining time for the current Block.

Volts/cm Displays the set voltage gradient for the current Block for 3 sec-

onds then displays the actual current (in mA) for the current Block.

Included Angle Displays the set included angle for the current Block for 3 seconds,

then displays the actual current (in mA) for the current Block.

Actual Current Displays the actual current for the Block in progress. The indicator

light should be on during a run. If the power supply is at current limit (500 mA maximum), the light will flash.

Pause/Start Run Initially, this starts the program and the indicator light will be lit.

While the program is running, pressing this key will put the program into PAUSE and the light will flash. Pressing the key again will restart the program.

In a multi-block program it is possible to examine the run parameters of any Block that is not currently being displayed by pausing the CHEF-DR III system. While in PAUSE, the RAISE and LOWER keys are active to scroll through any of the three Blocks. Any parameter in a Block may be displayed by pressing the appropriate key. Editing of run parameters is not possible once the program has started.

Program Termination

The program in progress may be manually terminated by holding down PAUSE/START RUN for 3– 4 seconds. A program can be terminated only while it is in the run mode; it can not be terminated in PAUSE. When the program is terminated two beeps will sound, and the right display will show OFF. Pressing PAUSE/START RUN again will start the program from the beginning.

When the program terminates under the timer control, the PAUSE/START RUN light will go off, it will sound two beeps per second for 5 seconds, and the right display will show OFF. 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 PAUSE/START RUN.

Clearing the Program

All parameters in Blocks 1, 2, and 3, can be cleared simultaneously to the default settings when the program is stopped or off. Press RAISE and LOWER on the right side of the panel for 5 seconds (it will sound 2 beeps per second).

Power Disruption

The CHEF-DR III system has a battery backed-up memory RAM that retains the current program if the power is interrupted. If the program was in progress (not in PAUSE) when the power went down, the program will automatically resume after 2 minutes in PAUSE mode after power is restored. The PAUSE/START RUN light will flash during this 2 minutes.

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 during isolation. To prevent breakage of large DNA molecules, intact cells embedded in agarose are lysed and deproteinized in situ. The agarose matrix protects the embedded DNA from shear forces and provides an easy way to manipulate samples. Processed agarose plug-DNA inserts are loaded directly into sample wells of agarose electrophoresis gels.

The most important and difficult task in preparing cells for imbedding in agarose is to obtain the proper cell concentration. Although optical density is frequently used, it is not reliable. Different cell lines or 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 greatly leading to over and/or under loading of the sample. To eliminate the need to generate a growth curve for each strain, a hemocytometer provides the most reproducible method for achieving the proper cell concentration for different types of mammalian, bacterial, yeast, or fungal cells. 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, and 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 is achieved using a thin well comb (0.75 mm).

3.3 Preparation of Agarose Embedded Mammalian DNA

The buffers, enzymes, and agarose in the following procedure are provided in the CHEF Mammalian Genomic DNA Plug Kit (catalog number 170-3591; see Section 9 for 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 use. 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 used.

2. Prepare a 2% low melt agarose (2% CleanCut™agarose is recommended, catalog num-

ber 170-3594) solution in sterile water and melt using a microwave. Equilibrate the solu-

tion 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.

4. Combine the cell suspension with an equal volume of 2% CleanCut agarose and mix gen-

tly but thoroughly. This results in a final concentration of 1% agarose. Keeping the

cell/agarose mixture at 50 °C, transfer the mixture to plug molds using sterile transfer

pipettes (Bio-Rad’s disposable transfer pipettes, catalog number 223-9524, are recom-

mended). Allow the agarose to solidify. This step can be expedited by placing the molds

at 4 °C for 10–15 minutes. This also adds strength to the agarose for removal from the

mold.

5. Using a 50 ml conical centrifuge tube, add 5 ml of Proteinase K Reaction Buffer

(100 mM EDTA, pH 8.0, 0.2% sodium deoxycholate, 1% sodium lauryl sarcosine,

1 mg/ml Proteinase K) for each ml of agarose plugs (e.g. use 25 ml of Proteinase K

Reaction Buffer for 5 ml of agarose plugs). Push the solidified agarose plugs, using the

snap off tool provided on the plug mold, into the 50 ml centrifuge tube containing the

Proteinase K solution. Incubate the plugs overnight at 50 °C without agitation.

Note: various cell lines have been incubated up to 4 days in Proteinase K without detri-

mental effects to the quality of DNA.

6. Wash the plugs four times in 50 ml of wash buffer (20 mM Tris, pH 8.0, 50 mM EDTA),

30 minutes to 1 hour each at room temperature with gentle agitation. If the plugs are to

be used in subsequent enzymatic reactions, it is advisable to wash the plugs in 1 mM

PMSF during the second or third wash to inactivate any residual Proteinase K activity.

7. Store the plugs at 4 °C. The plugs are stable for 3 months to 1 year.

8. Maintain the plugs in 1x Wash Buffer for long term storage. However, for subsequent

restriction digestion, the EDTA concentration must be lowered. Wash the plugs to be

restricted for 30 minutes in 0.1x wash buffer or TE. See Section 3.6 for information on

restriction digestion of plugs.

3.4 Preparation of Agarose Embedded Bacterial DNA

The buffers, enzymes, and agarose in the following procedure are provided in the CHEF Bacterial Genomic DNA Plug Kit (catalog number 170-3592; see Section 9 for information).

1. Inoculate a bacterial culture into 50 ml of LB Broth or appropriate media and grow with

agitation to an O.D.

600

of 0.8–1.0 at the appropriate temperature.

2. When the desired O.D.

600

is reached, add chloramphenicol to a final concentration of

180 µg/ml and continue incubation up to 1 hour while performing step 3.

Note: Chloramphenicol is used to synchronize ongoing rounds of chromosomal replica-

tion and inhibit further rounds of replication. This step is optional, but regions near the

replication terminus might be under represented. In addition, chloramphenicol will alter

the morphology of the cells over time, causing the appearance of a mixed culture, there-

fore proceed as quickly as possible with step 3.

3. Make a twenty-fold dilution of the above bacterial suspension using 10 µl bacteria, 20 µl Gram

Crystal Violet, and 170 µl saline or PBS. Place a small amount of the bacterial suspension on

a hemocytometer and count at 400x power. See Section 3.7 for hemocytometer use.

4. Prepare a 2% low melt agarose (2% CleanCut agarose is recommended, catalog number

170-3594) solution using sterile water and melt using a microwave. Equilibrate the solu-

tion to 50 °C in a water bath.

5. Remove 5 x 108cells for each ml of agarose plugs to be made. Centrifuge for 3 minutes

in a microcentrifuge. If the volume is too large, spin at 10,000 x g for 5 minutes at

4 °C in an appropriate size tube. Resuspend the cells in one-half the final volume of plugs

to be 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.

Caution: Some bacteria may be sensitive to the concentration of EDTA or the osmotic

strength of cell suspension buffer resulting in premature lysis of the bacteria. This pre-

mature lysis will result in DNA that is unacceptable for PFGE. Bacteria such as

Enterococci require 1 M NaCl in the buffer to prevent osmotic imbalance resulting in

lysis. Pseudomonasis sensitive to EDTA concentration, and dilution of the buffer may be

necessary. Most bacteria require no alteration of the buffer, but as stated in the above

procedure, mixing and imbedding of the bacteria should proceed as quickly as possible.

6. Combine the cell suspension with an equal volume of 2% CleanCut agarose and mix gen-

tly but thoroughly. This results in a final concentration of 1% agarose. Keeping the

cell/agarose mixture at 50 °C, transfer the mixture to plug molds using sterile transfer

pipettes (Bio-Rad’s disposable transfer pipettes catalog number 223-9524 are recom-

mended). Allow the agarose to solidify. This step can be expedited by placing the molds

at 4 °C for 10–15 minutes. It also adds strength to the agarose for removal from the mold.

7. Using a 50 ml conical centrifuge tube, add 5 ml of lysozyme buffer (10 mM Tris, pH 7.2,

50 mM NaCl, 0.2% sodium deoxycholate, 0.5% sodium lauryl sarcosine, 1 mg/ml

lysozyme) for each ml of agarose plugs, (e.g. use 25 ml of lysozyme buffer for 5 ml of

agarose plugs). Push the solidified agarose plugs, using the snap off tool provided on the

plug mold, into the 50 ml centrifuge tube containing the lysozyme buffer. Incubate the

plugs 30 minutes to 1 hour at 37 °C without agitation.

Note: Bacteria such as Staphylococcus aureus and some others are insensitive to lysozyme,

therefore lysostaphin must be substituted for lysozyme buffer. Additionally, adding

lysostaphin to the cell suspension immediately prior to embedding with agarose produces

high quality S. aureus plugs.

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