I want to perform a site-directed mutagenesis using non-overlapping primers (without 5‘ phosphate group). Does anyone know why it doesn’t seem to be necessary according to some protocols to perform the ligation of the staggered-nicked dsDNA PCR-product prior to transformation? some kits contain a ligase and DnpI for template degradation and others only DnpI but no ligase. thank you all in advance
Dear Marc
In my experience, no ligase is required for site directed mutagenesis. This linkage is easier than the cloning of one GOI since the overlapping regions formed by PCR are longer respect the one created from restriction enzimies, and there is only site to ligate and it is done directly from the bacterial host.
To undestand better the mechanism you can read the papers on the following links, about PIPE cloning, which is a cloning approach that i like a lot, which work quite well also for gene cloning and do not need any ligases but just specific e.coli strains that are more efficient in do in cell ligation.
https://pubmed.ncbi.nlm.nih.gov/18988020/
https://pubmed.ncbi.nlm.nih.gov/18004753/
if you are interested you can find some more information about it also in my simple scientfic blog ProteoCool (https://proteocool.blogspot.com/) in the Gene cloning section
good luck
Manuele
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