5' overhang on PCR primers

Eric Anderson e-anderson at ski.mskcc.org
Tue Jan 28 12:16:00 EST 1997


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
dropped it down to just the 6xHis-Tag and a Met (now just 21 extra bp) it
worked a little bit better but not that great.  if you're going to give it
a shot, i'd try using a proofreading polymerase (Pwo/Pfu) along with the
Taq.  when i finally got it to work it was with KlenTaq-LA (which i don't
know if you can get anymore).

one way to do this (if you can make oligos relatively cheaply) is to
attach a restriction site to the end of your oligo then make two
complementary oligos that have the epitope tag along with an overhang that
will ligate to your restriction site.  hybridize the oligos, gel purify
then ligate to your PCR product.

good luck and i hope this helped a little bit.

eric



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