May 6, 1990
Jim Howe
Explanation:
Pulsed field gel electrophoresis allows one to separate DNA molecules that range in size from <20 kilobases to 10 megabases. Conventional electrophoretic technique can only be used to separate molecules up to 30-50 kb in size, making long range physical mapping difficult, if not impossible. The basic problem with conventional electrophoresis is that large DNA molecules align to the electric field and travel horizontally through the agarose matrix of the gel. Thus, large DNA molecules will get caught up in the matrix and will not move much further than the wells in which they were loaded. The CHEF DR unit administers its electric field as "pulses" which are delivered from a hexagonal array of electrodes; this creates essentially two fields , one moving NW to SE (channel A), and the other from NE to SW (channel B), which intersect at 120 degrees angles. The result is that large molecules will travel in direction A for a short time and eventually get caught in the agarose matrix, but as the channel is changed to B, will realign with this field and move in this direction for a certain distance. The net movement (if the pulse times of A and B are equal) will be in the forward direction.
Components of the System:
1. CHEF DR II Chamber: This is the electrophoresis box itself, with the hexagonal electrode array. It is filled with 2-3 liters of 0.5X TBE buffer per run, which is recirculated and cooled during the entire run. The top of the box must be positioned properly before voltage will be delivered from the power supply to the electrodes. Gels are placed in the center of the unit within the white tabs.
2. Pulsewave 760 Switcher: This unit controls the switching intervals between channel A and B, the running time, and whether the switching interval remains constant during the run or it changes. The off/on switch is located on the right side, in the lower right corner.
3. Model 200/2.0 Power Supply: This controls the voltage delivered over the run. The maximum voltage delivered is 200V. The on/off switch is located as in the Switcher.
4. CHEF DR-II Drive Module: This is the interface of all the components of the system. More practically, it harbors the on/off switch for the buffer recirculating pump in its upper left hand corner, and its own on/off switch in the back upper left corner. This unit must be on for the other units to operate.
5. Hotpack Refrigerator/Circulator: This unit cools the 0.5X TBE buffer used during the run, necessitated by the high voltages used in these gels. It is composed of a tank which contains anti-freeze, within which the tubing running to the buffer pump and from the electrophoresis box is immersed. The temperature in the bath can be read on the thermometer on the superior surface of the "chiller", which should be at 4 degrees C; a dial on the front panel should be set between 0-4 degrees C to achieve this. There are two switches at the far left on the front panel (on/off; cooling) which should be turned on 2-3 hours ahead of each run to adequately chill the anti-freeze.
6. Pump: This circulates the buffer to the back of the chamber, across the box, out the front of the chamber to the chiller, then back to the chamber again. The speed of flow is adjusted by two clamps on the tubing located near the back of the chamber. Flow should be high enough to obtain good recirculation (monitored by the thermometer in the chambers front right corner which should be between 10-14 degrees C), but not be so vigorous as to force the back edge of the gel from the bottom of the chamber.
Planning a Run:
One must first decide what size fragments need to be separated by electrophoresis.
Different switching intervals will optimize separation of different sized fragments. This can also be influenced by the agarose concentration of the gel, the time of the run, and the voltage. Some examples given from the DR-II manual are:
A good approximation of pulse time can be arrived at by:
size of fragments to separate(kb) = Pulse time (seconds)
10
For example, yeast artificial chromosomes from the Center from Genetics in Medicine range from 100 kb to 1000 kb in size, and you wish to run a gel to determine the size of a new YAC:
100-1000 = 10-100 seconds
10
One can use a ramped switching time, that is, to begin at 10 seconds and gradually increase the pulse time to 100 seconds during the run, or one may wish to run at 60 seconds for 2/3 of the run then 90 seconds for 1/3 of the run, or one can run at a constant 50 seconds during the entire run. The first two yield similar results; ramping appears to have a slight tendency to compress the fragments in the central region of the gel, but optimizes separation of a wider variety of fragments. When the fragments are all of similar size, a constant pulse time seems to give the best result. The voltages and run times given in the table are good guides to setting these parameters. In general, let the size of the larger fragments you wish to separate determine the parameters (be aware that much smaller fragments may run off gel).
Pouring a Gel:
Determine the optimal agarose concentration, and dissolve agarose in 0.5X TBE in microwave or by autoclaving (Many people get superior results by using highly purified agarose, e. g., SeaPlaque low melting temperature agarose). Allow the agarose to cool in a 50 degrees C bath. Clean the gel casting stand and set comb height. Pour the gel and immerse the comb such that the plastic screw behind the wells abuts the back wall of the casting stand, and all the wells are parallel to the back wall. Allow to set for 30 minutes, then pull out comb.
Loading a Gel:
One can load either DNA plugs or liquid DNA with dye/stop mix added to it. For plugs, take a 5 mm slice and gently place into a well on the tip of a flat spatula. Push plug down into well with another spatula till it touches the bottom. Be sure not to crush plugs, for these will run poorly, and distinct bands may be lost. When all plugs are loaded, then pipette 200-300 ul of agarose (can use the remainder from the bottle you used to prepare gel with) over each plug until the wells are full. Allow 5-10 minutes to solidify, then place in the CHEF DR-II chamber. It is helpful to circulate the buffer for 1 hour before the run in order to bring the buffer to running temperature.
If liquid samples are to be loaded, add enough stop mix to bring to approximately 1.2X (1X solutions may float out of well). After the gel is placed in the chamber and immersed under buffer, turn pump off, and load as with any other gel (except pipette tip ends should be cut to minimize shearing of DNA). Replace cover, and begin run; turn pump back on after 5-10 minutes.
Setting Run Parameters:
Pulsewave Switcher:
1. Initial A Time. Push and then hold down raise/lower buttons until LED displays initial pulse time desired.
2. Final A Time. Push and then hold down raise/lower buttons until LED displays final pulse time desired; if the final and initial times are the same, then this run will have a constant pulse time. If they are different, then there will be a ramped switching interval if there are no changes in the mode. If they are different, and one wishes to have two different pulse times, then one will need to change the mode setting.
3. Start Ratio. This should always be set at 1.0, which means that the channel A pulse time will be the same as the channel B pulse time; however, if one desires to run a field inversion gel (FIGE), this will need to be adjusted upward.
4. Run Time. Push and then hold down raise/lower buttons until LED displays the total run time desired; if one is using 2 alternating pulse times, one needs to set the different run times for each pulse time before setting mode.
5. Mode. This determines type of switching interval. Constant or ramped runs do not need to be adjusted from the default value of 1. For two alternating pulse times, enter all the parameters of the first pulse time, then by pressing the buttons for initial A time and run time simultaneously, set the mode to 10 by using raise/lower keys. Then enter the parameters for the second pulse time, and set to mode 11. Note: At the end of a run, a buzzer will sound until the Pulsewave switcher is turned off.
Power Supply: After the pulse times are set, turn on this box (if turned on earlier may start delivering when initial run parameters are being set). Push volts button, then adjust to desired level using raise/lower buttons.
At end of run:
Turn off power supply and pulsewave switcher; turn off pump, remove chamber cover and carefully lift gel out of box and place into gel box. We have used plastic kitchen spatulas for this task to minimize the chances of gel breakage; this is especially a problem when gels are of low-melt agarose. Place large beaker under the short tubing at the front of the gel chamber, remove clip, and allow buffer to drain. Run pump for short time to clear tubing of buffer, then tilt chamber slightly to drain. Replace clip, then be sure to turn off Drive Module and Refrigerator/Circulator.
Southern Transfer of Pulsed-Field Gels:
1. At the end of the run, place the gel in 1 liter of dH2O containing 20 ul of 10 mg/ml ethidium bromide and gently rock for 30 minutes. Photograph the gel as per routine.
2. Rinse briefly in ddH2O, then soak in 0.25N HCl for 30 minutes to depurinate the DNA.
3. Rinse briefly in ddH2O. Denature in 1.5M NaCl, 0.5N NaOH, 30 minutes with agitation.
4. Rinse briefly in ddH2O, then place on a stack of blot block with one sheet of 3MM Whatman paper. Carefully remove all the bubbles by rolling with a test tube, then place nylon membrane over it. Place another layer of Whatman over this, followed by 6 blot blocks, paper towels, and a weight. Use 1.5M NaCl, 0.25N NaOH as the transfer buffer. Allow to transfer overnight, with frequent changes of buffer.
References:
Instruction Manual to BioRad CHEF DR-II Pulsed Field Gel Electrophoresis System, Richmond, CA, 1988.
Instruction Manual to BioRad Pulsewave 760 Switcher, Richmond, CA, 1988.
Instruction Manual to BioRad Model 200/2.0 Constant Voltage Power Supply, Richmond, CA, 1988.