What do you think is the best mutagenesis strategy if you want to mutate several - (Sep/17/2007 )
What do you think is the best mutagenesis strategy if you want to mutate several nt's in your insert?
I will toot my own horn here and suggest that you take a look at the paper I published in NAR a few years ago.
Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h
Better DNA sequencing traces
Decided to go with QuikChange Multi site directed mutagenesis kit from stratagene. Will test it tomorrow hopefully :-)
Interesting Daniel! Have probably not read it carefully enough but what to do if you have more than two mutations?
It depends where the mutations are located. If they are within 50bp of each other it is very easy (you just incorporate them into both long primers). If they are far apart then you need to do it as a multiple step process. While this may seem slower you gain the advantage of been able to check the mutations as you go along. I have tried introducing multiple mutations using multiple primers with the quick change system and never had much luck - this was one of the reasons I developed the SLIM method.
DNA sequencing software