site directed mutagenesis
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Hi,
I am trying to make some site directed mutants of protein I am working on. I have successfully created some mutants. I am using mixture of Taq and Pfx polymerase. I usually do amplification for 4 min for 20 cycles of my 3.5kb plasmid. I get bright band after PCR and get them gel purified and treat with DpnI. After transformation I get a lot of colonies, but unfortunately after lot of screening I am not getting my desired mutation. I only get my wild type gene, which s supposed to be not there after DpnI digestion. Can any body help.
Thanks
I am trying to make some site directed mutants of protein I am working on. I have successfully created some mutants. I am using mixture of Taq and Pfx polymerase. I usually do amplification for 4 min for 20 cycles of my 3.5kb plasmid. I get bright band after PCR and get them gel purified and treat with DpnI. After transformation I get a lot of colonies, but unfortunately after lot of screening I am not getting my desired mutation. I only get my wild type gene, which s supposed to be not there after DpnI digestion. Can any body help.
Thanks
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