Human and Clinical Genetics (LUMC) - LabJ_Protocols©

Denaturing Gradient Gel-Electrophoresis

(Rolf Vossen; last modified April 28, 2001)

Abbreviations

Purpose

The following protocol is used for preparation and electrophoresis of a gel for Denaturing Gradient Gel-Electrophoresis (DGGE). The protocol is based on using the Biorad DCODE gel electrophoresis system.

Protocol

GEL PREPARATION

  1. fill the electrophoresis tank with electrophoresis buffer (~7 l), place the lid of the system on the tank, switch on the system and set the temperature to 58oC
  2. place a clean and dry gradient-mixer on a magnetic stirrer; make sure that the junction tubing and the tubing that leads to the cassette are closed
    NOTE: the height-level of the gradient-mixer is very important and should be ~32 cm; a different level will have a dramatic effect on the gradient formed
    NOTE: different results are obtained when different gradient mixers are used. In our hands, using a home-made gradient mixer with cylinders that have a 2 cm inner diameter, we obtained better results than with some commercially available mixers
  3. clean the spacers and glass plates with soap, rinse thoroughly with water, clean with ethanol and air-dry
  4. assemble the gel cassette and place the cassette on the gel casting device
  5. take 1.0 ml 9% acrylamide solution, add 25 ul 10% APS and 1.5 ul TEMED, mix and pour in to the gel cassette
    NOTE: a plug will be formed to prevent leakage when the gradient-gel will be cast
  6. cast the gradient-gel: freshly prepare 12 ml of each of the two desired acrylamide/UF solution mixes
    EXAMPLE: to prepare a gel with a 15%-55% UF-gradient, the following mixes are made:
    1. 15% mix: 1.8 ml 100% acrylamide/UF solution + 10.2 ml 9% acrylamide solution
    2. 55% mix: 6.6 ml 100% acrylamide/UF solution + 5.4 ml 9% acrylamide solution

    NOTE: depending on the fragments to be analysed, gels with a 20%-60% or 35%-75% gradient can also be used

  7. add to each mix 65 ul 10% APS and 3.3 ul TEMED, mix gently and pour the low percentage mix in the left cylinder of the gradient-mixer. Open the junction-tubing carefully until just a few drops enter the other cylinder. Fill the right cylinder with the high percentage mix and switch on the magnetic stirrer. Open the junction-tubing and the tubing that leads to the cassette simultaneously, the gel cassette will be slowly filled
    NOTE: from the moment the gel solidifies, let it polymerise for at least 1 hr
  8. drain off excess fluid from the top of the gel
  9. prepare the stacking-gel solution, carefully pour the stacking-solution on top of the gradient-gel and insert the gel comb (polymerisation takes about 15 min)

GEL LOADING AND ELECTROPHORESIS

  1. carefully remove the gel comb and rinse the slots with water
  2. switch of the DCODE-system and carefully remove the lid to place it on the stand
  3. put the gel(s) in the gel holder and place the holder in the electrophoresis tank
  4. fill the upper buffer compartment with electrophoresis buffer
  5. mix 15 ul PCR-product with 3 ul 6x loading-mix
  6. rinse the slots again with buffer and carefully load the samples
  7. place the lid back on the electrophoresis tank and switch on the system; the run is performed 16 hr at 80 V

GEL STAINING AND ANALYSIS

  1. disassemble the gel cassette and carefully remove one glass plate
  2. while attached to the other glassplate, put the gel in a tray with 0.5 l EB-staining solution, place the tray on a tumbling-plateau and stain for 15 min
  3. destain the gel 5 min in water
  4. analyse the gel on a UV-transilluminator

Material

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