Pulse Field gel Electrophoresis

  • In 1982, Schwartz introduced the concept that DNA molecules larger than 50 kb can be separated by using two alternating electric fields
  • Pulse field gel electrophoresis is normally used to done for study the clonality among microorganisms. This is a is a technique used for the separation of large DNA molecules by applying  periodic electric field from different directions that changes periodically to a gel matrix.
  • Larger DNA more than  15-20kb migrating through a gel essentially moves together in a size-independent manner, the standard gel electrophoresis technique was unable to separate very large molecules of DNA effectively which led to the practice of pulsed field gel electrophoresis.

 

General Protocol for Pulse Field Gel Electrophoresis

 

 

This Protocol was standardized by Dr Avinash Singh by using a 3- day protocol (Grundmann et al., 2002).

 

DAY 1

  1. Pick a single colony and inoculate into 5 ml BHIB.
  2. Incubate the culture overnight at 37 °C.

DAY 2

  1. Vortex overnight culture tubes gently to re-suspend cells.
  2. Transfer 600µl of the culture into a 1.5 ml microtube.
  3. Centrifuge at 13,000 rpm for 1minute.
  4. Aspirate the supernatant from the tube, and re-suspend cells thoroughly into

500 µl TE  by vortex.

  1. Ensure that the pellet is resuspended completely.
  2. Following Centrifugation, re-suspend cells into 250µl TE by vortex, and put the tube on ice.
  3. Add 10µl of 1 mg/ml lysostaphin.
  4. Dissolve 2 % Low Melting Point (LMP) Agarose (Bio-Rad) into autoclaved

de-ionised water.

  1. Add 250 µl agarose into microtube.
  2. Mix very gently and briefly by pipeting.
  3. Immediately pipette agarose/cell mixture into plug molds (2 block per isolate)
  4. Leave molds on ice for 10-15 min.
  5. Add 1,000 µl Lysis buffer to microtube and pre-warm at 37 °C.
  6. Dispense plugs into the appropriate 1.5 ml microtubes.
  7. Ensure that two plugs are in each microtube and that Lysis buffer covers

the plugs.

  1. Incubate for 60 min at 37 °C.
  2. Add 100ul Proteinase K (10 mg/ml in autoclaved water Incubate micro tube at 50 °C overnight.

DAY 3

Digestion of DNA with Restriction Enzyme

  1. Trim plug to 2 mm x 3 mm size and place in appropriate 1.5 ml microtube.
  2. Use a cover slip to keep the right shape.
  3. Add 200 µl PMSF in TE for 30 min. at 50 °C.
  4. Aspirate PMSF in TE buffer.
  5. Ensure that pipette does not damage plugs through these steps.
  6. Wash plugs 3 times in 200µl TE buffer.
  7. Remove TE buffer thoroughly from tubes.
  8. Prepare restriction enzyme (RE) Master Mix:

Per Sample

  1. Sterile distilled water 85 µl
  2. SmaI buffer (x 10 conc.) 10µl
  3. BSA 01µl
  4. SmaI (10 Units/μl) 04µl

…………………

TOTAL  100µl

  1. Add 100 µl RE Master Mix to each microtube.
  2. Ensure plug is covered by RE Master Mix.
  3. Incubate microtube at 30 °C for 3 hours
  4. The plug can be stored at 4 °C up to 2 overnights, if RE Master Mix is
  5. Replaced with TE (10 mM, 1 mM; pH 8.0) buffer.

Prepare Agarose Gel.

  1. Add distilled water, x10 TBE and PFGE grade agarose to 250 ml conical flask:
  2. Distilled Water x 10 TBE PFGE grade agarose
  3. For 15 wells gel 95 ml 5 ml 1.0 g

 

Prepare Electrophoresis Buffer and loading plugs into Gel

  1. Prepare 1,600 ml x 0.5 TBE buffer.
  2. Adjust the Gel Chamber on level surface.
  3. Pour 800 ml x 0.5 TBE buffer and then start to circulate by pump.
  4. Wash Gel Chamber with 1,000 ml distilled water with circulation, before

Pouring  TBE buffer.

  1. The water should be drained thoroughly after washing.
  2. Switch on CHEF-DRII Drive Module.
  3. Switch on pump and adjust pump flow rate to 20
  4. Cool x 0.5 TBE buffer to 14 °C.
  5. Switch on Chiller Unit.
  6. Set Chiller to maintain temperature of 14 °C.
  7. Ensure buffer is circulating, check for air bubbles and blockages.
  8. Slant slightly the whole chamber.
  9. Place an approx. 30 mm thick book or plate under the distal end of the

chamber.

  1. Place gel onto the middle of Gel Chamber.
  2. Load digested plugs and molecular weight standards (lambda and/or NCTC
  3. 8325) into well.

Electrophoresis

  1. Cool TBE buffer and gel sufficiently to 14 °C before starting electrophoresis.
  2. Run gel under the following condition,

1st set                                               2nd set

Run time     10 hours                       13 hours

Initial switch 5.0 sec                         15.0 sec

Final switch 15.0 sec                        60.0 sec

Voltage 200 V                                       200 V

 

  1. After electrophoresis run is completed, switch off all equipment:
  • CHEF-DRII Switcher
  • Pump
  • Chiller Unit
  • Switch off all electric sockets.
  1. Open lid of Gel Chamber and remove gel with using support.
    • Remove carefully so as not to damage electrodes and gel.
    • Drain Gel Chamber.
    • Wash Gel Chamber.
    • Use 1,500 ml distilled water with circulation by pump.
    • Wash at least 1 min.
    • Re-drain gel chamber.

Gel Staining and image storing

  1. Stain gel for 30 min. in ethidium bromide (1 μg/ml).
  • Gloves MUST BE WORN when handing ethidium bromide.
  1. De-stain for 45 min. in distilled water.

View under transilluminator and photograph using gel documentation system (Bio-rad)

  1. Gel analysis was done by Gel compare II 6.6 (Bioneumerix) version software.
Pulse Field Gel Electrophoresis
Flow chart for Performing Pulse field Gel Electrophoresis

Protocol for different microorganisms needs some modifications in buffers and instrument settings (for details please contact Dr Avinash Singh.https://www.researchgate.net/profile/Avinash_Singh3)