5' overhang on PCR primers

Alex Brands abbrands at artsci.wustl.edu
Thu Jan 30 18:12:00 EST 1997

On Tue, 28 Jan 1997, Eric Anderson wrote:
> In article <32EE3BDB.5A6B at watson.princeton.edu>,
> tvogt at watson.princeton.edu wrote:
> > I want to do PCR with a primer that has a 5'overhang of 
> > 50bp of unpaired nucleotides for the creation of an 
> > epitope tag. CAN this be done?  Is there a maximum number 
> > of nucleotides that can be left unpaired and PCR still 
> > work well.
> tom,
> my personal experience is that it can't really be done.  i tried to attach
> a 6xHis-tag plus a Factor X cleavage site (total about 45 bp extra but i
> don't really remember) that way and i got very miserable results.  when i

I'm not really sure why Eric had trouble, but many yeast labs routinely
make knockout/knockin constructs via PCR, and for that, the primers each 
have at least 45 bases on the 5' end that don't match the original
template.  Of course, they will match the new template generated after the
first couple rounds.  As long as there are enough annealing bases on the
3' end (18 for the yeast KO constructs) I can't imagine why it would be a

Alex Brands
Washington University

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