I need to substitute at three positions of a structure. In order to inactivate the active sites. I will be substituting Serine, Aspartic acid and Lysine sites with Alanine.
There are many kits available to do this and there is a lot of information available with a simple internet search. A good place to start would be
https://www.neb.com/applications/cloning-and-synthetic-biology/site-directed-mutagenesis
There is a modified protocol proved much more efficiency than the most of conventional ones https://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-8-91 and the attached lab protocol you might find much easier to follow.
Hi Wardatul,
To perform site-directed mutagenesis upon a plasmid, I usually design a pair of primers which are complementary to each other that have the codon you wish to mutate located in the middle. I make sure there are approximately 15 nucleotides either side of the mutant codon in my primers, to ensure that they will bind to the original plasmid sequence. When you design the primers, try to change the least number of nucleotides as possible, as this will increase the efficiency of your mutagenesis. If you want to create more than one amino acid substitution in the same plasmid, you might need to design separate primers and do the mutagenesis one after another, not all together if your chosen amino acids are separate.
Once I've ordered these primers and they've been synthesised for me (no modifications needed), I then use a thermocycler to amplify my plasmid - make sure you use a long extension time so that the DNA polymerase has enough time to go all the way around your plasmid (e.g. 10 minutes). Once complete, I digest the PCR products with the Dpn1 restriction enzyme, which will destroy the original plasmid DNA template because it is methylated, leaving you with only your new mutant plasmid; run an agarose gel after this to double check that you do have PCR products and then you can transform some into E.coli DH5 alpha cells. Pick a few colonies, grow each of them overnight in 5-10 ml of LB (+ your antibiotic of choice), miniprep and then sequence to check the mutation has been created.
I have attached my protocol for you to view.
Best of luck with your work!
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