I am trying to create blunt ends to fit the fragment in different plasmid vector which doesn't have exact RE sites. I know this reactions are theoretically easy to consider but practically very tricky ..!! Hence, I would like to have suggestion from experienced experts @ ResearchGate...!! :)
Thank you.
Hello Tushar,
I don't think there are any big tricks with both protocols. Both enzymes are active in large range of conditions. For Klenow your just need to supplement restriction buffer with 33uM each dNTP. Here is working protocols form NEB; NEB enzymes are usually the best choice too:
https://www.neb.com/protocols/1/01/01/removal-of-single-stranded-extension
https://www.neb.com/protocols/2014/01/13/protocol-for-blunting-ends-by-3-overhang-removal-and-fill-in-of-3-recessed-5-overhang-ends-using
Good luck!
Personally I prefer T4 polymerase, e.g. https://www.neb.com/products/m0203-t4-dna-polymerase
Yoram
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