PFGE Protocol
DO NOT PUBLISH WITHOUT PERMISSION OF AUTHORS
K. Eric Wommack
1. Make plugs – see plug-making protocol. Make sure there is at least 2.5 liters of 0.5x TBE already made.
2. Pour PFGE gel
a. Make a 1% Biorad PFGE agarose gel in 0.5x TBE – for the large gel rig, the buffer volume is 200mL, so use 2g agarose (the small rig holds 100-150ml).
b. Set-up gel rig - remember when putting in the comb to use microscope slides to ensure that the comb sits evenly along its entire length
c. Pour in warm agarose and cool
d. Cover with with 0.5x TBE (to prevent it from drying) and saran wrap (easiest in large pyrex dish). Place at 4oC for at least 20min, preferably overnight.
3. Turn on the power for the Chef PFGE control unit.
4. Pour in 2.2L of cold 0.5x TBE to the PFGE rig (make sure it’s been cleaned properly since the last use).
5. Turn on the pump (about 80/min is a good rate). Once the buffer is flowing, turn on the chiller.
**Important! - Do not run the chiller without running the pump, or the buffer may freeze in the lines. If this happens, turn off the pump and chiller and let the buffer thaw prior to re-starting the pump.
6. Set the temperature on the chiller and allow to equilibrate up while you load the gel.
7. Loading the gel:
a. Cut the appropriate number of plugs (about 2mm wide) from the λ ladder – same number as ladder lanes on the gel.
b. Prepare the Hind III digested λ ladder if needed – for 3 lanes, mix 40uL of ladder with 8uL of 6x loading dye and load 12uL in a lane.
c. Remove the comb slowly and carefully.Load your ladder and samples into the gel wells.
d. If you will be plugging your wells with agarose, load the liquid ladder before putting the gel in the rig, heat and melt 1.5% InCert agarose, and using a 1mL pipette, fill wells with agarose. Allow to cool and set,
e. If you will not be plugging your wells, do not load the liquid ladder samples, skip step 7d and proceed to #8.
8. Set up gel in PFGE rig
a. Make sure buffer temperature is at gel running temperature.
b. Remove the gel from the pouring rig, and remove any extraneous gel from the bottom and edges with your hands.
c. Place the gel in the PFGE rig. Make sure it is seated securely inside the frame all the way on the bottom of the rig.
d. If loading liquid samples:
i. turn off buffer chiller and pump briefly
ii. load the samples
iii. restart the pump and chiller.
9. Close the PFGE rig lid.
10. On the panel for the main PFGE unit, press the desired angle set-up, then input your run conditions as asked for.
11. When the run is done, press the number required to clear the program and stop the run
12. Turn off the chiller
13. Turn off the pump.
14. Spike the SYBR Gold bath with 25uL of stock SYBR gold, whisk with a pipette to evenly distribute the stain.
15. Lift out the gel on the plate and carefully slide the gel into the SYBR bath. Gently rock the bath and gel for 20-30 min, then remove the gel from the bath, place in de-stain, and scan on Typhoon imager.
16. Meanwhile, clean the the PFGE rig.
a. Drain the buffer into the 10 L carboy labeled for “PFGE waste”.
b. Pour in 2L of DI water, turn the pump on, run for 5 minutes before draining
c. Repeat for a total of 3 DI rinses.
d. After the third rinse, drain all lines, leave the lid open and the rig tilted up to continue to drain and dry.
Turn off the main power to the PFGE control unit.
