PCR with genomic DNA.

1x reaction

2 ul of genomic DNA (50-100 ng/ul)

2.0 ul 10X Taq polymerase buffer

0.5 ul Forward primer (20 pmole)

0.5 ul Reverse primer (20 pmole)

2 .0ul dNTPs (2 mM each)

0.2 ul Taq polymerase (1 unit)

12.8 ul deionized water

Total: 20 ul

8x reaction

16 ul 10X Taq polymerase buffer

4 ul Forward primer (20 pmole)

4 ul Reverse primer (20 pmole)

16 ul dNTPs (2 mM each)

1.6 ul Taq polymerase (1 unit)

103 ul deionized water

Add 18 ul of reaction mix to 2 ul of genomic DNA.

PCR conditions:

5min - 94˚C

10 cycles of touchdown PCR with 0.5˚C decrease every cycle

20 sec - 94˚C

20 sec - 63˚C (decrease by 0.5C every cycle)

2 min - 72 ˚C

35 cycles of

20 sec - 94˚C

20 sec - 58˚C

2 min - 72˚C

72˚C - 10 min (to add A overhang for TA subcloning)

4˚C – standby

Take 3 ul of PCR product to check the quality of PCR products on 1% agarose gel.