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do you put two pairs primers in one tube when you do site mutations? - (Feb/12/2008 )

Hi, I want to do two site mutations on one plasmid. can I put two pairs primers in one PCR reaction ? if not, how would you suggest to proceed?
e.g. after one round of PCR, purify the product ( or not) and use it for second round of PCR?


I haven't done 2 mutations in 1 plasmid. I guess, I would do mutation transform it and then grow it up and then go for the other. it might take time but then you are sure that it worked.


You could try the multi-site-directed mutagenesis kit from stratagene, designed to introduce more than 1 mutation. But if you were only to do it once, I would go as Scolix suggested.