protocol colony PCR?

[Bas Jansen]

In article <7r7u75$m51$1 at info.service.rug.nl>, "Rene Haanstra"
<r.haanstra<nospam>@biol.rug.nl> wrote:

> Dear Netters,
> 
> Does anyone have a protocol for colony PCR that works for most
> bacteria? I experience a lot of problems when i try to do a 16S PCR
> on colonies of bacteria. Two out of five colonies don't produce a product.
> It seems to be not a problem in breaking the cells, since we get a product
> with the same colonies when we perform a REP-PCR. In REPs the product
> does not serve as a template, so the cells are broken. We think that the
> DMSO in the REPs helps accessing the DNA, but we don't like to use DMSO
> because it also has an effect on the annealing temperature and we don't
> like to optimize the annealing temperature again.

Your post suggests that you've already optimized your 16S PCR. My
question is: with which template? Pure genomic DNA? Pure plasmid DNA?
If so, you definitely need to re-optimize your PCR when using
not-so-pure DNA.

All the best,
Bas

-- 
bjhj at dds.nl