How to make a point mutation in a wild type plasmid. - (Oct/04/2005 )

Hi, all:

My lab has already constructed a plasmid containing the gene of interest. Now, I'm contemplating to make some point mutations in the gene so as to investigate the interaction of proteins.

I know I should design a primer containing a mismatched base, but I have another concern. If my primer with a mismatch works well in the PCR amplification, how can I know that the amplified &mutated gene is the correct size? I'm thinking about to add two endonuclease restriction site, in doing this, I can simply cut the mutated plasmid and run it on agarose gel. Hopefully, I will be able to see two bands with correct size.

any ideas of how I can add two restriction sites flanking the gene without disturbing its expression?

or do you think my thinking is wrong? if so, please correct me as of how to get to my aim.

thanks

The easiest way to create point mutations in a plasmid is QuickChange Site-Directed Mutegenesis.
You can buy a kit or just primers and pfu-polymerase.
http://www.stratagene.com/manuals/200518.pdf

Yeah -- that's the kit. I knew I used a site-directed mutagenisis kit once several years ago, but couldn't recall whether it was from Stratagene or Promega...