PCR

[Andrew Doherty]

Usha RAMAN wrote:
> 
> Hi
> 
> I am trying to design primers (20-mer, both forward and reverse) close to
> the cloning site in the original TA cloning vector. But they include a EcoR1
> and BstX1 (forward) and EcoRV and EcoR1 (reverse) site. Will these
> sites pose a problem during PCR. Any suggestions will be helpful.
> 
> Thanks
> 
> Usha
> raman at email.chop.edu

A question first - are the restriction sites in the vector or primers?
If they are in the vector, then this will not cause a problem in the
PCR. As far as the reaction itself is concerned, a DNA sequence is a DNA
sequence. Whatever restriction sites are coded for in that sequence is
irrelevant. If they are in the primers, then make sure that you have
12-15 bases of complementary sequence at the 3' end of each primer.

But why are you amplifying from a TA vector? Surely you've already
amplified a product and cloned it to have it in the TA vector in the
first place???

Hope it helps

Andy D
-- 
*************************************************************
Dr Andrew Doherty		email -  a.doherty at bris.ac.uk
Dept. Anatomy			Tel (0117)9287421
School of Medical Sciences	Fax (0117)9287402
University of Bristol
University Walk
Bristol UK
BS8 1TD
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