PCR PRIMER PROBLEM
ziegler at ifi.med.uni-muenchen.de
Mon Jan 30 11:21:43 EST 1995
I am trying to amplify mRNA from cells that have been transfected with
reporter plasmids. The downstream primer is spanning a 600 bp intron.
There are 4 nucleotides upstream and 17 downstream of the intron. Somehow
this primer manages to anneal to the plasmid DNA and to get extended by
Taq. Does anybody have an explanation for that phenomenon? I´ve been
thinking about exonucleases chopping off the 4 nucleotides upstream of the
intron(i.e. 3´end of the primer). Do you think a circular 5kb plasmid
would loop out 600 bases just to anneal to lousy 4 nucleotides?
Interestingly, I´ve been able to increase specificity by lowering the
magnesium concentration to 0.8 - 1.2 mM. Unfortunately, the system is not
very stable and each reverse transcription and therefore differing
template concentrations require different a magnesium concentration...
I hope somebody can help me. Thanks, Stefan
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