PCR protocol

• 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:
    • 5 minutes at 95°C (especially important if using colony pick or unopened cells af any form)
    •  
    • 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)
    •  
    • 6 minutes at 72°C
    
    
    

    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 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