Design Tagged Primers

Kunika kunika_jain at rediffmail.com
Thu Jan 17 01:17:18 EST 2002


Hello All,

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?

Any suggestions or ideas would be highly appreciated. 

Thanks in advance. 

Regards,

Kunika



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