Hi everyone,
When introducing point mutations in a plasmid using site directed mutagenesis, can you insert more than one at the same time (in the primer) in case they are close to each other? Or is it more effective to introduce one mutation per reaction?
Hi Sofia,
Yes you can introduce more than one mutation in the plasmid which are close to each other simultaneously using site directed mutagenesis. We have performed it several times in our lab using the Agilent Technologies QuikChange II kit and it has worked great each time. But make sure that you use the XL version of the kit if your plasmid along with the gene of interest is more that 8kb in size.
Happy sciencing!
Jagaree
Thank you both for answers.
However, do you know roughly how far apart these mutations can be in order to be classed as close enough to perform direct site mutagenesis with the same primer?
Cheers,
Sofia
Hi Sofia,
The closest mutation we have done is two amio acids i.e- six nucleotides apart. I am positive that you can mutate consecutive amino acids as well using the same primer. While designing mutant primers select codons as close to the original sequence as possible. You should be good that way.
Good luck!
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