PCR troubleshooting (fwd)

Terese M Barta-1 barta001 at STAFF.TC.UMN.EDU
Tue Jul 6 14:17:17 EST 1993


This is one of two replies I received regarding the trouble I am having
with PCR. Although I know my DNA is not degraded, the information
below may be of help to others at some time:

(T. Barta, U of MN)

---------- Forwarded message ----------
Date: Fri, 2 Jul 1993 21:38:45 GMT
From: Andres Ferber <andres at calvin.jci.tju.edu>
To: barta001 at staff.tc.umn.edu
Cc: andres at calvin.jci.tju.edu
Subject: PCR troubleshooting

I think you should consider the possibility that your genomic DNA is 
degraded.
If you excluded this possibility,then there is a trick that sometimes works 
for mamalian DNA.  Try to use 10% glycerol(final concentration) in your PCR 
rection.
I do not think that RNA can interfere with DNA amplification.  I did not 
address that question specifically but I have some indirect evidence. Namely 
when I do Rt-PCR to amplify RNA and there is some DNA contaminating my RNA 
preparation I amplify such DNA .I know the amplification product comes from 
DNA because I use primers that span an intron and therefore the 
amplification product is bigger and because I also get the product when I do 
not do the reverse transcription part of the procedure which then excludes 
that the amplification comes from hnRNAs.In this particular condition there 
is "a lot of RNA" and probably few DNA templates and nevertheless I can 
amplify them (unfortunately!!). All this in mamalian cells extracts...I 
think for the purpose of the question that it is not important.

Andres
andres at calvin.jci.tju.edu





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