PCR help

who? SMITHLM at IRAD.AFRC.AC.UK
Tue Jul 13 13:53:00 EST 1993



I have a slight problem which I thought somebody may be able to help me with.
 I am currently trying to use PCR to detect viral oncogenes,and things were
going swimmingly - Myb and Mil succumbed to my charms, but then came Myc.

 After infecting CEFs with MC29, and extracting the DNA, I can't PCR the 
viral myc gene whatever I try. So far the list includes:
      Magnesium titration from 0 to 10mM
      Glycerol from 0 to 10%
      Formamide from 0 to 10%
      Restricting the genomic DNA with an enzyme which doesn't cut v-myc
      Pre-boiling the DNA for 10 minutes.


      Preboiling the DNA for 10 minutes.

 The primers I use are both 24mers and have a GC content of approx 50%.
However, the region I am trying to amplify has 67% GC - is this the 
problem, and if so how do I get around it.
 I know the genomic DNA I use is OK, because other primers will amplify it!
Any and all suggestions welcomed.

                          LEE SMITH
                      (e-mail: SMITHLM at UK.AC.AFRC.IRAD)



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