Hi,
I want to do an overlap extension PCR (OE-PCR) using a PCR-amplified plasmid backbone and a PCR-amplified insert. The OE-PCR shall add the insert into the plasmid similar to this protocol:
Article Overlap extension PCR cloning: A simple and reliable way to ...
I don't want to transform the plasmid (which now still contains two nicks) into E.coli or anything similar.
But I need to close the nicks of the plasmid in vitro. Do I need to add a Ligase and a Kinase to the reaction?
Or is there a Ligase which does not need 5' Phosphorylation of the DNA pieces?
Best,
Filip
Hey,
I haven't used this particular protocol (so take my answer with a grain of salt) but in general in such cases you want to phosphorylate your oligos prior to the first PCR (as at least the regular T4 PNK exhibits much higher phosphorylation efficiency on ss than ds substrates) and then, in the end, you can use the T4 DNA ligase for nick repair.
Best of luck!
Hey,
I haven't used this particular protocol (so take my answer with a grain of salt) but in general in such cases you want to phosphorylate your oligos prior to the first PCR (as at least the regular T4 PNK exhibits much higher phosphorylation efficiency on ss than ds substrates) and then, in the end, you can use the T4 DNA ligase for nick repair.
Best of luck!
Sounds like you would be pretty much ready to go to use a Gibson cloning mix like:https://www.neb.com/products/e5520-nebuilder-hifi-dna-assembly-cloning-kit#Product%20Information.
That way you don't need to worry about optimizing the enzyme conditions. I have used this in situations as you have described and have had 90% efficiency in recovered clones (NEB10beta cells) from inserts as small as 100bp to 8kb (total vector size from 5kb to 12kb). There are plenty of other Gibson or Gibson-like reaction mixes and most places would be willing to give you a free sample to try out.
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