PCR from yeast genomic DNA

Mon Jan 8 10:53:39 EST 1996

Just a few additional notes on this subject:

1.  Crude DNA extracts often contain PCR-inhibitory substances, so try a
dilution of any non-working prep before discarding.
2.  We find 99 C denaturation is unnecessary, and 94 C is quite adequate.

3.  Various methods for long PCR are marketed.  Most of these seem to simply be
a mix of 99% one thermostable polymerase and 1% another.  If you need to save
money, you can certainly mix your own.  We keep stocks of a half dozen
polymerases just for this purpose.  We also find that these mixes sometimes
yield usable product when standard methods fail for short PCR, so consider this
for your 1 kb products.

4.  Published methods for improving fidelity and length include shortening 
denaturation times and adding a tad of DMSO.  

5.  We concur with the previous posting that home made genomic DNA is quite
adequate for most purposes, as is home made RNA for RT-PCR.

Mike Leibowitz
Robert Wood Johnson Medical School

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