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
