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PCR—from (Dr. Chen, Univ. College London)
1) Add the following to a microfuge tube:
10 ul reaction buffer
1 ul 15 uM forward primer
1 ul 15 uM reverse primer
1 ul template DNA
5 ul 2 mM dNTP
8 ul 25 mM MgCl2 or MgSO4 (volume variable)
water (to make up to 100 ul)
2) Place tube in a thermocycler. Heat sample to 95C, then add 0.5 -1 ul of enzyme (Taq, Tli, Pfu etc.). Add a few drops of mineral oil.
3) Start the PCR cycles according the following schemes:
a) denaturation - 94C, 30-90 sec.
b) annealing - 55C (or -5C Tm), 0.5-2 min.
c) extension - 72C, 1 min. (time depends on length of PCR product and enzyme used)
repeat cycles 29 times
4) Add a final extension step of 5 min. to fill in any uncompleted polymerisation. Then cooled down to 4- 25C.