Mutagenesis again (View forum version)



moerae

Posted 05 August 2010 - 01:35 PM

Hi,

Tried my first mutagenesis reaction and well it was kind of a dud. I've followed the Stratagen QuikChange protocol... cleaned up the PCR reaction products, ligation reaction and DpnI digestion and transformed. I got nothing on my plates.... I have no idea if I did something wrong or not?

Chimp

Posted 09 August 2010 - 05:12 AM

Did you do include any kind of control? Like transforming with the original plasmid? It could be something as simple as your competent cells going bad. In my experience they are best fresh, especially if you make your own competent cells.

It could of course also be something with your PCR (e.g. badly designed primers).


Cheers!