I am trying to do site saturation mutagenesis in 5.2 kb plasmid which is having my desired gene . I have tried different conditions and enzymes also but it did not work. please help me. currently i am using Phusion HF DNA polymerase.
Hi Nisha,
Maybe one by one mutagenesis is a practical way. PCR followed by gibson assembly and transformation.
Good luck!
What is your protocol?
Overlap extension approach should work fine, for example. Both primers would carry the NNN somewhere in the middle (or only one of them in more tricky designs). Phusion HF is nearly the best on the market.
Try to design your primers and follow the protocol as described in the linked publication which has been proved more efficient (http://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-8-91).
I tried overlapping extension PCR by using mutagenic primers, having NNS in middle. But it did not work even i tried many conditions. I got colonies but when i submitted for sequencing i found no mutation at desired site. Please help me.
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