noncomplementary primers and pfu polymerase

PAtrick Lynch patrick.lynch at bbsrc.ac.uk
Wed Feb 6 16:08:23 EST 2002


Hi all,

Thanks to all the individuals who posted contributions to my query. In the
end, I did a magnesium titration and the PCR worked really well at 6mM and
not at all at the usual 2mM Mg2+. Furthermore, I had a similar PCR with a
5'-27 noncomplementary base tail but the amplicon was 1 kb in size. This,
too, only worked at an elevated Mg2+ conc BUT I had to increase the
elongation step to 2.5 min instead of the usual 1 min - special thanks to
the Frank Fackelmayer for mentioning this. Sorted!

Cheers

Patrick




> Hi, 
> 
> I'm trying to amplify a 0.5 kb fragment of DNA using a set of primers, one 
> of which has a 5'-27 base tail that isn't complementary to the initial 
> template, with pfu polymerase (cloned - Stratagene). Unfortunately, the
PCR
> never works. This problem of 5'-noncomplementary primer ends and 
> amplification with pfu has been reported before in this NG. I wonder if 
> anyone has ever succeeded in doing this sort of amplification with pfu? 
> 


<http://www.biowww.net/forum/read.php?f=1&i=5302&t=5302>




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