Touchdown PCR

[Paul N Hengen]

Joao Vasconcelos Costa (jcosta at pen.gulbenkian.pt) wrote:

: Don et al's original description of touchdown PCR, that was discussed
: some days ago in this group, delas with nondegenerate primers. Their
: primers had a Tm of 64, calculated by the 4GC+2AT rule. The starting
: annealing temperature was 65 and the final touchdown temperature was 55.

: How should these temperatures be set when using degenerate primers?
: In normal PCR or hybridizations with degenerate primers I usually
: consider, to be safe, the lowest Tm in the mixture of oligos. Should
: also be this the starting temperature in touchdown PCR? And the final?

There was a recent article in BioTechniques that described parameters used for
touchdown.  It might be worth reading:

@article{Hecker1996,
author = "K. H. Hecker
     and K. H. Roux",
title = "High and low annealing temperatures increase
both specificity and yield in touchdown and stepdown {PCR}",
journal = "BioTechniques",
volume = "20",
number = "3",
pages = "478-485",
year = "1996"}

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Paul N. Hengen, Ph.D. (pnh at ncifcrf.gov)
National Cancer Institute--FCRDC--Frederick, Maryland 21702-1201 USA
phone:(301) 846-5581 fax:(301) 846-5598
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