Design Tagged Primers

dbell 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 
part
: >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 
annealing
: 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).
: DK


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?

*~*~*~*~*~*~*
Deanne Bell
USDA Agricultural Research Service
9611 S. Riverbend Avenue
Parlier, CA 93648
(559) 596-2806
(559) 596-2791 fax
dbell at fresno.ars.usda.gov
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