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