skspoidn at reading.ac.uk skspoidn at reading.ac.uk
Fri Jun 4 09:35:51 EST 1993

I am not familiar with the Biorad mutagenesis kit, because as much as I hate 
to admit it, I use a lab protocol devoid of kitstuff. However, places where 
U DNA synthesis has fallen down and been picked up again in our lab include...

1...Too high an inoculum of phage used in the initial preparation of UDNA,
 resulting in carry over of wt DNA

2...Too much polymerase added in the synthesis step. This can realy muck up
 the reaction, including kicking off your mutagenized strand and synthesizing 
a wt strand. Use only as much pol as suggested in the kit, or even less.

3...Use g32 (AKA SS DNA binding) protein for a several fold increase in synthesis
 efficiency, during the initial primer binding step.

Our full protocol sent direct, to save bandwidth.

Mike Poidinger
Dept of Microbiology
University of Reading

More information about the Methods mailing list