PCR to make labelled probes

ANDY PHILLIPS andy.phillips at afrc.ac.uk
Tue Feb 1 03:57:27 EST 1994


Micheal Frohlich suggests using 'hot' PCR with degenerate primers to
make probes for screening libraries. In our experience, it is unwise
to assume that the products of such a reaction are 100% the intended
target. Using degenerate primers on genomic and cDNA followed by plasmid
cloning and sequencing we have found between 2% and 100% of the 'correct' 
products in the amplified pool. These individual clones can then be 
labelled for screening, by PCR if you wish.

Anyway, thanks for the 32P-PCR recipe. We haven't used PCR for probe-
making up to now, because of Taq's high Km for dNTPs.

Andy Phillips



More information about the Methods mailing list