I have a gene of interest (HIV-1 envelope) inserted into a plasmid. Now I want to insert specify nucleotide substitution in some point by site directed mutagenesis. Please suggest me some good commercial kit/protocol and of course your experience.
To be honest, you dont need a kit, you just have to have appropriate primer pair, PCR materials, DpnI RE, E.coli host to amplify DNA that s all! if you wanna have a protocol I can send you!
I agree with Ananda but if you want to try a kit, QuickChange site directed mutagenesis kit from AccessAgilent would be best. You can also go for a cheaper option and can try KOD hot start DNA polymerase kit. I have used both of them successfully.
I agree that you don't need to use a kit if you want to cut down on costs. But if you're in a hurry, I've the Phusion Site-Directed Mutagenesis Kit from Thermo Scientific before which worked pretty easy.
Can you please share the protocol with me? Please mail me at [email protected].
I agree with the past comments. You essentially need to design good primers (I generally use Quickchange primer design. or the PrimerX primer design. You can optimise the quickchange mutagenesis protocol (adjusting the concentration of the template and/or adjusting the PCR conditions. All the best!
http://www.genomics.agilent.com/primerDesignProgram.jsp?&_requestid=388519
http://www.bioinformatics.org/primerx/cgi-bin/DNA_1.cgi
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