PCRprotocol

• PCR reaction

  Protocol for 50μl reaction - adjust amounts if necessary, for a 20μl reaction use the same volumes of primer and dNTP-mix, but adjust the volume of water and 10x buffer, for a 100μl reaction use twice the volume of everything. This protocol is for with 10x buffer with added MgCl2, if this is not included add it to a concentration of 1.5 mM or other concentration found to be optimal.

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.

• 5 minutes at 95°C (especially important if using colony pick or unopened cells af any form)

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• 30 times:

• 30 seconds at 95°C (depending on PCR machine)

• 30 seconds at 50°C (adjust according to annealing temperatures of primers)

• 30 seconds at 72°C (long PCR products (>1kb) require longer)

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• 6 minutes at 72°C

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 60?C.

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