2 replies to this topic

[hpsauce14]

Hi there,
            I was wondering if anyone can offer any advice on the best method for mutagenesis experiments, which I am yet to embark on. I am looking at substituting 3amino acids with alanines at one site and 4 amino acids (substituting again with alanines) at another site, simultaenously. The gene in question is a 10.9kb gene encoding a cellwall anchored protein (with LPXTG motif) from Streptoccus gordonii. Is this gene too big for accurate and successful mutagenesis? If so, I could just mutate the 4kb N-terminus (TheC-terminus is 6.9kb and made up of 14 imperfect repeat blocks.) I have had alook through the posts and quite a few people are having problems with the kits (stratgene etc.) Is there another way other then bought kits? Also are there any 'hot tips' for mutating my monster gene.

Thanks!

Helen

[Warren]

hpsauce14, on Oct 19 2009, 03:44 PM, said:

Hi there,
            I was wondering if anyone can offer any advice on the best method for mutagenesis experiments, which I am yet to embark on. I am looking at substituting 3amino acids with alanines at one site and 4 amino acids (substituting again with alanines) at another site, simultaenously. The gene in question is a 10.9kb gene encoding a cellwall anchored protein (with LPXTG motif) from Streptoccus gordonii. Is this gene too big for accurate and successful mutagenesis? If so, I could just mutate the 4kb N-terminus (TheC-terminus is 6.9kb and made up of 14 imperfect repeat blocks.) I have had alook through the posts and quite a few people are having problems with the kits (stratgene etc.) Is there another way other then bought kits? Also are there any 'hot tips' for mutating my monster gene.

Thanks!

Helen

Yeah you can just use PCR.  I would just make subclones containing smaller fragments and do the mutagenesis on them, then subclone back into the full-length.  It might sound like more work, but its really not if you can find convenient restriction sites, plus you can sequence your whole PCR-generated sequence from the subclone (alot easier than sequencing an entire 11 kb region to assure no unwanted mutations!)  Warren..

[AlfaBeta]

You even don't need kits. There are a lot of non-commercial sytems, that are very efficient and simple. You can try them. I.e. megaprimer method, or MALS mutagenesis e.t.c.  w.ncbi.nlm.nih.gov/pmc/articles/PMC2759926/

Best wishes