Is it possible to induce site-directed substitution mutation by quick-change method on linear dsDNA? or it has to be cloned in some vector?
If yes, should it be treated with the Dpn1 enzyme after mutagenesis?
There are site directed mutagenesis protocols that would work fine on linear DNA but then of course you would need to clone after the mutagenesis. The typical quick-change method however would not be suitable (on it's own) since it would not generate intact molecules for cloning.
I never thought about it, but certainly it could work. Sometimes the fragments are small enough that ordering the long oligos is the fastest way possible, followed by Sanger sequencing.
I don't think it work.
If you use PCR product as template for site-directed mutagenesis I don't think this DNA will be methylated (contrary to plasmid extract from almost all E.Coli strains). Thus, the parental template will not be digested by DpnI.
Thank you for your answer. Now, it's clear to me. Could you please suggest the best suitable method to induce site-directed mutagenesis on linear DNA. My product size is around 600 bps and mutation will be induced 30th bp from 3'end.
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