I am working on mutagenesis recently and I need to mutate glumamine to cysteine for the labeling reaction. According to their codons, I need to change three nucleotides. I prefer to put the mutation in the middle of one of my primers, which are around 20-30 nucleotides long. I am wondering what is the best way to do so. Could I do it in a reaction? Or mutate two points first, then another one point?
I have personally used up to 4 mutations (4 nt) in the middle of a 25bp primer without any trouble, so go for it in one step !
You can always increase the primer lenght. Usually, 16-18 matched bp are enough, even if you have several unmatched bases as well. A popular example is primers with restriction sites, which often have 16 matched bases, then up to 8 additionnal nt.
Hi,
thsi will work, if you need some more background you could also look for overlap extension mutagenesis. This helps if you mutagenesis is in the middle of the sequence or you don't want to circular amplify your plasmid
Sven
Dear Cheng
you can easly do it using Pipe cloning approach.
you can dind infomation about it on my blog: ProteoCool (https://proteocool.blogspot.com/)
and on
https://link.springer.com/protocol/10.1007%2F978-1-59745-196-3_6
good luck
Manuele
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