Design Tagged Primers
dbell at qnis.net
Thu Jan 17 13:36:16 EST 2002
On Wednesday, January 16, 2002 10:36 PM, D.K.
[SMTP:dk at no.email.thankstospam.net] wrote:
: kunika_jain at rediffmail.com wrote:
: >Hello All,
: >In order to calculate the Tm of the primers shall I consider just the
: >sequence specific part of primer or complete primer (sequence specific
: >of primer + tag sequence).
: Just do it. In any case, you must have primers with a given sequence
: (tag+ORF) so who cares what Tm calculation or secondary structure
: tells you? If it is ditry, gel purify, if it does not work, change
: temperature. The steps will not be different from those you'd do if
: you have "perfect" primers (as anyone will tell you, those are not
: guaranteed to work from first time either).
IMHO I agree with the "just do it" attitude. Most of the TM calculators I
have used do not give me very accurate annealing tmps when compared to
optimization experiments. Do you have access to a gradient thermocycler?
USDA Agricultural Research Service
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dbell at fresno.ars.usda.gov
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