Hi All, I want to generate a mutation in plasmid, but my plasmid is around 16 kb, is it possible to do it? I am planning to design two non-overlapping primers containing the mutation site to PCR the whole plasmid. Thank you!
Yes it can be done, using standard quick change primers designed as if they were to deal with a 5 kb plasmid, you just need much longer elongation times in your PCR programme. This however, doesn't mean you should do it, unless you haveaccess to a free sequencing service.
In my lab we usually do the point mutagenesis in small plasmids and then transfer the sequenced verified region to the bigger expression vectors. Even with 5kb plasmids, you can never be sure if besides the desired point-mutation you also got some other unknown mutation in the plasmid backbone. There is only one way to find out, sequence the whole plasmid, and because subcloning is a routine that takes only 3 days of less than half time work, we tend to subclone the mutagenised coding region after verification by sequencing. We have done quick-change on +15kb plasmids as well, but rather than sequencing the entire 15 kb we just subcloned the verified region into another 15kb plasmid so we didn't have to worry about the plasmid backbone and all the relevant reporter genes, control genes and selectable markers that are found on the backbone and that we need for our experiments.
:)
It will be safe to take a small fragment of 2-3 kb out form the larger 15 kb and then relegate back after confirmation of desired mutation. Otherwise you have to sequence the full length fragment to confirm the authenticity of the native sequence.
Hi there,
Yes, its certainly possible.
I would suggest you to use 4 primer with spacing 4 kb distance to improve the success of the SDM PCR.
Rajkumar
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