colony pcr question

Don Walthers gene_jockey26 at yahoo.com
Mon Jan 10 17:47:25 EST 2000


I routinely perform PCR on colonies of M. xanthus (67% G+C). Take a large
colony and resuspend in 10-20 ul H2O. Test 1-10 ul of template. I usually do
95-5' (95-1', 50-2' and 72-X') X30, with and without 5% DMSO. The low and
lengthy annealing temp has been a critical factor in getting these GC-rich
PCRs to work. Also the difference between 94 and 95 in the denaturing step
is important. Make sure your Taq doesn't mind 95. We use standard BRL Taq.

--
Don Walthers
B.S.Microbiology U of Illinois at UC, 1996
Ph.D. Microbiology U of Idaho, 2001?
gene_jockey26 at yahoo.com






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