>I can observe the amplified PCR product before and after DpnI digestion
>(see image in http://ompldr.org/vY2x3aw), but cannot get any colony on LB
>plates. I'm using very fresh super competent cells so that I've got dozens
>of colonies with 60 ng of the parental/non-mutated vector as positive control.
Sounds like you don't have competent enough cells. 60 ng should give
millions, not dozens of colonies. (Decently competent cells are 10^5/ng of
plasmid). Only a tiny proportion of the observed polymerase product in
QuickChange reactions is actually transforming, so you have to assume that
you start with a lot less than 1 ng. So if you are not seeing at least a
1,000 of colonies after 1 ng plasmid transformation, chances that you will
have positive clones in your QCh transformation are pretty low.
- Dima
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