Hi !
Recently, i have did some site directed mutate reaction. However, all the six PCR reaction product have some problem demonstrated by sequencing, the mutated sequence have one inserted region just after the mutated site, and the inserted region is exactly the primer, sometime the primer repeated twice, and sometime repeated three times. So, my question is that, which factor lead this, the primer i designed? the enzyme i used ( primerstar premix)? or the template i used ? additionally, it often appear in my PCR reaction experiment.
thanks advance!
Primer design. It has to be corrected at primer design step. Also make sure you use an enzyme which has proof reading activity.
Yeah I agree with Goodwin, sounds like a primer design issue. You can always try a site-directed mutagenesis kit; I've used them in the past with pretty good success. Several companies make them and I can personally attest to NEB's.
I have actually have had this problem and while primer design may help it may not solve the problem either. I think its a combination of the sequence context of the target site, the primer sequence, and the pcr cycling parameters. I actually have asked this question before and did not receive any clear reason as to why this may occur (old question below). At the time I was using q5 from NEB and actually spoke to them at a product show and the amount of primer and template you use can result in some weird insertions. They suggested using more template and less primer and to screen several colonies. I ended up using this suggestion and screened about 10 colonies, of which only 2 came back with the proper mutation and no insertions. Hopefully this helps, but more than anything I would just try a few changes and screen many mutants.
https://www.researchgate.net/post/What_is_a_possible_explanation_for_multiple_primer_insertions_during_site-directed_mutagenesis
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