How to control the PCR where genomic DNA as a template

[Dr GR Taylor]

gina_briscoe at cellbio.duke.edu (G. Briscoe) wrote:
>
> I want to fish out a new gene from bacteria  by PCR where genomic DNA was
> used as a template and degenerated primers were applyed. I dont know if I
> need predigest geomic DNA before PCR. If so or not, how to control the
> raction. Thanks.

You shouldn't need to digest bacterial genomic DNA, you could cut between your 
primer binding sites.

Control reaction by annealing temp primarily (for degenerate primers
you may need to start as low as 45 degree C, increasing until
you get specific products), also Mg conc (usually around 1.5mM) conc 
and primer conc (usually around 25-50uM).

Beware, single bands many not be the gene you were looking for,
only sequencing will confirm.

Good luck

Graham Taylor
Regional DNA Lab, Leeds, UK
e-mail gtaylor at hgmp.mrc.ac.uk