iam at mail.boku.ac.at
Mon Jun 26 08:44:34 EST 1995
I´ve been trying to establish a PCR protocol that uses the oligo dT 5´-ends and
oligo dG 3´-ends of (homopol. tailed) cDNA for cloning (with C(14)-EcoRI and
T(18)-NotI primers respectively). In the beginning it did not work at all.
Only very short garbage was amplified. But after some playing around
with (template),(Mg++) etc. I encounter a different but (for a PCR rookie)
even more disturbing situation:
I get a smear from 300bp up to more than 23kb.
How is it possible to get PCR products that long from ordinary cDNA?
Do I see unspliced primary transcripts?
Or is it possible the templates form oligomers (tandems or the like)?
My extension time is 3min (at 72). I anneal for 2min at 62deg.
Does Taq really have the time to go that far on a single template?
Any suggestions? Georg
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