I tried to do site directed mutagenesis several times I used takara enzyme to do the reaction
I did the digestion with DpnI and I also use negative control (using the template with and digested it with DpnI )and did the transformation. With the negative control I got no colony which means the DpnI worked pretty well and I got several colonies with the reaction mixture.
so I purified the plasmid after I grew the bacteria in LB medium.
I sequence the plasmid but I did not get the desired point mutation.
the resulting sequence was exactly as the template ???
what should I do to optimize or fix this problem?
There is a modified method has been proved much more efficient than the commercialized one ( http://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-8-91). It explains the causes of low efficiency of the original method and shows how to make your mutagesis more efficient. The attached lab protocol is summarized based on that paper which might be easier to follow.
but I already bought the primers sets
and all of them primer-primer complementary
how could I use them and at the same time improve the effeciency of PCR reaction
Hi there...
I guess, there is an issue of primer design. The point mutation site must be flanked by at least 15 nucleotides on both sides to have better efficiency during the PCR amplifications or else everything looks fine as per your explanations.
the primer length was 36 nucleotides and the changed nucleotide was in the middle?!
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