Running Manual (not BaseStation) Acrylamide Gels

 

  1. Put bottom tray into gel rig.
  2. Remove clamps and wrap.
  3. Remove boot and wash acrylamide off glass with normal water.
  4. With gentle water running take out comb.
  5. Remove any extra acrylamide and dry glass.
  6. Take 2 black gel spacers (little and sponge-like)
  7. Put plates into gel rig with short plate in back and sponges on side right on top of the plates (to prevent leaks).
  8. Lock plates in with side clamps of gel rig. Clamp down very tightly.
  9. Close drain on side.
  10. Add 1X TBE to top, filling just a little below back knob just above liquid.
  11. Fill bottom tray until little knobs next to plate are just above the liquid.
  12. Prerun gel for 20 minutes at 1900V, 50W.
  13. For 10ul PCR reactions, add 1:1 loading dye (so 10ul of loading dye). Put the dye in a tray and use a multi-channel pipet. Pour the extra back into the dye container.
  14. Heat plate for 3 minutes at 95C using Fisher hotplate and put weight on top.
  15. The ladder (often use 400bp) is in 4C fridge and it needs to be heated to 95C for 2 minutes.
  16. With a syringe, rinse the urea out of top of gel.
  17. Put comb (teeth down) back into gel until teeth have entered acrylamide but not too deeply.
  18. Add 3 ul of sample to each well using an 8 channel pipettor if possible.
  19. Attach power leads and run at 1900V, 50W until dye front is near bottom.
  20. Turn off power and open drain to remove buffer from top chamber.
  21. Remove glass plates from gel rig.
  22. Push spacer on right out a little bit and take a spatula to carefully open plate and remove smaller plate. Set it aside.
  23. Put 0.8 grams of agarose into 30 ml nanopure water.
  24. In a 50ml conical tube, add 25ml of nanopure water and 0.5ul of SYBR Green.
  25. Pour SYBR Green/water solution into melted agarose flask.

[alternatively add 50ml nanopure to 0.8g agarose and microwave; add 0.5ul of SYBR Green]

  1. Mix together and pour over gel. Smooth solution out over gel with hands quickly.
  2. Let it stain for ~10 minutes and then wipe plate with Kimwipe to remove any acrylamide from bottom and sides of plate so acrylamide won’t fall in Flourimager.
  3. Put into Flourimager with combs facing towards you.
  4. In flourimager program: Templates -> Acryl top -> name scan something
  5. After scanning, clean gel off plate with a paper towel.
  6. Use Sigma cleaner and a sponge to clean plates.
  7. Rinse 1X with regular water and 1X with dI water. Can reuse 1X TBE until it turns blue.