PCR : Smearing problems

Jon Chappel drjon at clone.scripps.edu
Fri May 12 18:11:46 EST 1995


In article <JO_C.1.2FB18D3C at vcp.monash.edu.au>, JO_C at vcp.monash.edu.au (Jo
Caine) wrote:

I have spent the last 2 years on and off trying to PCR from a variety of
sources. Firstly I should say "do not despair" as the key to successful
PCR is certainly perseverance. Secondly I would ignore anyone who believes
blindly in the Tm approximations most people end up with. Try annealing
temps all the way up to (and including) 72 degrees. One of my PCR
reactions only worked if I cycled between 94 and 72!! Thirdly if you get
contaminants of higher molecular weight than you want then force the
amplification of the shorter one by reducing the extension time (even down
to as low as 15 seconds). Using more template often adds to my PCR
problems. Finally I found that one of my PCR contaminants I saw when
amplifying from a cDNA library I had made was contaminating 3'primer form
the cDNA first strand synthesis carried over as a contaminant in the PCR
reaction. cDNA spin columns solved that problem..!

I hope this solves some of your queries as I sweated blood to find them
out for myself.
--Jon
  
> PROBLEM 1:
> 
> I am screening a quick clone cDNA library using PCR. .......
> 
> .......Anymore ideas?
> 
> Thank you in advance!!
> 
> All members of the biochemistry lab present at mailing.
> 
> E mail : Jo_C at vcp.monash.edu.au



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