Design Tagged Primers
dk at no.email.thankstospam.net
Thu Jan 17 01:36:12 EST 2002
kunika_jain at rediffmail.com wrote:
>For expression cloning I need to amplify ORFs. To amplify the ORFs I have
>designed sequence specific primers tagged with His tags and some other
>similar tags like, att recombination sites and GST tag.
>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).
>If I use a tagged sequence specific primer and consider only the sequence
>specific part for primer for Tm calculation then from the 2nd cycle of PCR
>when the complete primer (Sequence specific+tag ) will bind to the template
>the Tm for that will be different.
>And if I consider complete primer (Sequence specific+tag ) for primer Tm,
>then Tm will not be appropriate for very first cycle of PCR.
>Please help me how should I proceed and which Tm should I consider.
>Also if primer is tagged then what I think is it should be analyzed for
>secondary structures. Am I correct?
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).
More information about the Methods