URGENT...site directed mutagenesis - (Sep/25/2001 )
can anyone help me out withthis..i am trying to do a site directed mutagenesis using stratagene kit (which i myself have reconstituted)but inspite of getting quite anumber of colonies ...everytime a sequencing is done it is revealed that it is the wild type....can anyone help me out as to whatthe problem might be and also whether there is anyother method to screen so many colonies without sequencing if there is norestriction enzyme site at the site where the mutagenesis is to be done...moreover since the pcr is for abt 15 cycles or so icannot run it on a gel to see if the pcr has worked
I have use Stratagene Quikchange with little problem. I would assume that you have check that all the oligos are correct. Try a test reaction, i.e. one without oligos and see if you still get colonies. Your problem suggests to me that the restriction digest step is not working properly, therefore check this step carefully. You should expect that all or nearly all of the native plasmids will be destroyed. In my experience all the ones I have sequenced are mutants. One thing you should know though, Quikchange does not work well with plasmids larger than 10k. It may be useful to try other enzymes instead of pfu if your plasmid is large.
thanx for the suggestion. i shall check it out.
I am having a problem with the Stratagene site directed mutagenesis kit. Basically the oligos are designed from the Stratagene website and PAGE purified. All the other components of the reaction mix (i.e. templates, dNTPs,water, Reaction buffer, Enzyme) work fine, with other oligos, which are admittedly half the length of the mutant oligos. Its just when I put in the mutant oligos into the reaction mix, I see no amplification.
Is there something I can do to fix that?