PCR:Template-oligomers?

David Clayton dclayton at uiuc.edu
Thu Jun 29 23:59:06 EST 1995


In article <3smdk2$4mb at mail.boku.ac.at>, iam at mail.boku.ac.at (Georg
Seifert) wrote:

> Hi Netters
> 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 


I'm no expert myself, but could it be that the problem is more
fundamental?  Several factors can retard the migration of DNA in a gel to
produce a smear...high salt, etc.  Have you tried cleaning your PCR
product before running it on a gel?

Rick



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