PCR Trouble -Reply

JENKINS at PCIMSG.PCI.UPMC.EDU JENKINS at PCIMSG.PCI.UPMC.EDU
Fri Jun 3 14:39:24 EST 1994


>I have been trying to amplify a region of human genomic DNA.  I
>have  amplified it successfully in the past, but, under the same
>conditions,  some new samples are not working.  I have done a
>phenol/CHCl3 extraction  and ethanol precipitation to clean them up,
>run the reactions with MgCl2  concentrations ranging from 0.5 mM to
>4.0 mM, and tried increasing the  amount of DNA.  I got a very faint
>band when I increased the amount of  DNA to 400ng (50 ul rxn).  I
>figure it must be something about the  samples because I have run
>positive controls, and they always look great.
>Does anyone have any advice on what I can do?

We have found that exchanging the buffer the DNA is dissovled in with
a centricon 100 helps a great deal.  We do not change the buffer, just
replace it with fresh.  Apparantly this gets rid of small contaminants
that interfere with the PCR reactions.  
Hope this helps

Frank J. Jenkins, Ph.D.
University of Pittsburgh





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