help with site-directed mutagenesis - What polymerase and cells to use? (Oct/16/2007 )
I need to make three amino acid conversions in a gene.I was looking at the Quickchange kit from Stratagene. I was hoping to use the procedure in the kit without actually buying it. In our lab, we have Dpn1 enzyme and Pwo polymerase. Does anyone know if Pwo polymerase is non-strand displacing (ie works with ds DNA) like the pfuturbo in the kit? Also, are TOP10 or DH5alpha cells eligible for blue white selection?
Is there any other strategy to do site directed mutagenesis?
I would appreciate any help from people who have done this before.
We did not buy the kit, only the Pfu enzyme from stratagene for mutagenesis. It works. We got DpnI from NEB and used DH5-alpha cells.
I'm suppose to make 2 amino acid changes in a gene. I've cloned this gene in puc18 and then I've confirmed that by sequencing. Now I should do site directed mutagenesis by changing codons.
First of all do I have to do plasmid prep to make those changes? the next step is the designing the primer for my mutagenesis. How can I design these primers??(The PCR product size is around 1.5 Kb)
after making those changes how can I return this fragment to the wild type??
I appreciate if you give e details if you got time.
The online program for site-directed mutagenesis primer design is PrimerX (http://www.bioinformatics.org/primerx/index.htm). It makes it so easy!