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Buffer Gradient Sequencing Gels

  1. Wash new plates thoroughly using detergent and dry. Wipe each plate with 100% ethanol. Wipe the plate with ears only using Rain-X to siliconize.
  2. Clamp plates together, remembering to place a little vacuum grease between side and bottom spacers, and place spatula to spread plates slightly at the top to facilitate pouring the gel.
  3. To pour your gel you need:
    - 100 ml measuring cylinder
    - TEMED
    - 25% APS
    - 2 bottles for pouring
    - P200
    - 2.5 x TBE sequencing buffer
    - 0.5 x TBE sequencing buffer
    - 35ml pipette and bulb

For a 60cm buffer gradient gel:

  1. To one of the bottles add 74 mls of 0.5 X TBE gel buffer.
  2. Using the same measuring cylinder, add 21 mls of 2.5 X TBE gel buffer to the second bottle.
    - To "a" add 130ul of 25% APS and 85ul to "b". Swirl gently.
    - To "a" add 40ul of TEMED and 25 ul to "b". Swirl gently.
  1. Suck up 10ml of 0.5 x, then all of the 2.5 x in the pipette. Be careful not to mix the 0.5 x and 2.5 x in the pipette (this is to form a discontinuous gradient). Lift plates and slowly pipette this in. As it finishes lower plate so that a column of gel solution extends up the plate. Pour in remainder of 0.5 x solution, maintaining a continuous stream. An uninterrupted flow is important to avoid bubbles and prevent the gradient being displaced when the bulk of the 0.5 x flows in.
  2. Push in shark toothcomb about 4mm.
  3. When the gel is set remove clamps, bottom spacer and comb. Wash comb area and push teeth of comb into gel. Mark any areas that have bubbles so you don't load any samples in this region. Fill tanks with 0.5 x TBE.
  4. Remove air bubbles where the bottom spacer was, using a syringe and needle full of 0.5 x TBE running buffer. Flush comb out before loading the gel.
  5. Pre-run gel at 55W (constant power) for 15 minutes.
- 2.5 x TBE gel mix for sequencing
- 230 gms urea
- 62.5 mls 20 x TBE
- 75 mls 40% acrylamide (19:1 and without urea)
- 25 gms sucrose
- 25 mg Bromophenol blue
- DDW to 500 mls
- 0.5 x TBE gel mix for sequencing
- 460 gms urea
- 25 mls 20 x TBE
- 140 mls 40% acrylamide no urea
- DDW to 1 litre.
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail:
David Bowtell PMCI October 1998