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PCR

PCR protocol

  1. In a 200µl PCR tube take 5µl 10X PCR buffer (from enzyme kit).
  2. Add 1µl dNTP-mix.
  3. Add 1µl of 10 pmoles/µl solution of Forward primer.
  4. Add 1µl of 10 pmoles/µl solution of Reverse primer.
  5. Add 1µl template DNA, or a tiny bit of colony on agar-plate picked with a tooth-pick.
  6. Add 40.5µl (or 41.5µl if using a colony pick) dH2O.
  7. Add 0.5µl Taq DNA polymerase (5U/µl).
  8. Mix gently by pipetting.
  9. Place in PCR machine, and close hot-lid (if the machine does not have a hot-lid add a couple of drops of mineral oil on top of reaction to prevent evaporation.
  10. Perform PCR with a suitable program for the template, primers, and thermal cycler:

    Agarose gel for analysis of PCR products

    Protocol for 60 ml gel - adjust amounts if necessary.

  11. In a 250ml conical bottle take 0.72g of agarose.
  12. Add 60 ml of water.
  13. Heat to boiling in microvawe owen.
  14. Cool down to 60C.
  15. Add 2µl 10 mg/ml ethidium bromide.
  16. Pour gel in tray with tape at ends and 8 tooth comb inset.
  17. Allow to set.
  18. Take 10µl of the PCR reaction and place on a piece of Saran wrap.
  19. Add 2µl 6X loading buffer.
  20. Load on gel along with molecular weight marker.
  21. Run at 100V until dye front has reached approximately 3/4 down the gel.
  22. Inspect under UV.

Solutions

dNTP-mix for PCR
10µl 100mM dATP, 10µl 100mM dCTP, 10µl 100mM dGTP, 10µl 100mM dTTP, 60µl DEPC-treated dH2O
6X loading buffer for agarose gel
25µl 1% (w/v) bromo-phenol blue, 25µl 1% (w/v) xylene-cyanol FF, 30µl 100% glycerol, 20µl dH2O

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