14 May 2020 3 935 Report

Hello,

I need to change two adjacent nucleotides.

* I've added 5% DMSO

* I've used an annealing temp that is 5 degrees higher than the tm of the primers.

* Im using a hot start kit

But after sequencing is still get a long repeat of the primer in the site of mutagenesis.

Analysis show that the dimerization in due to nucleotides that are adjacent to the mutations, so I cant design a primer without them.

What else can I try?

Do you think its possible to use a long primer with the mutation flanking? A bit like Gibson reaction?

Thank you!

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