I work in a microbiology lab where we do a lot of cloning. I have always used restriction endonucleases to cleave the DNA to have sticky ends and not blunt ends. I currently am working on a project that suggests using a blunt end restriction enzyme, such as EvoRV, and then to perform a T-tailing of the plasmid, pl4440. My question is why do I not treat my pcr'd insert with a restriction digest just like I would the plasmid as the protocol suggests? How do I know the insert has ends that are able to ligate into the plasmid if I don't design the restriction sites into the primers?

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    $\begingroup$ If i understand the question correctly, your are asked to make a single EcoRV cut in your backbone plasmid and make blunt ended inserts? $\endgroup$ – Bez Oct 21 '14 at 19:23
  • $\begingroup$ Well, the plasmid has a single cut in the back bone but I'm not sure how the insert will ligate correctly if I do not treat the insert to make blunt ends. $\endgroup$ – IamAbiome Oct 21 '14 at 20:33
  • $\begingroup$ if then you can only use EcoRV and nothing else then you have to use blunt ended inserts but you will have problems with insert orientation and multiple ligations of insert! $\endgroup$ – Bez Oct 21 '14 at 20:35
  • $\begingroup$ I think you can turn sticky ends into blunt ends with klenow enzyme. Been too long to be sure. $\endgroup$ – user137 Oct 21 '14 at 22:32
  • $\begingroup$ @user137, yes thats correct! the OP can use DNA polymerase I, large (Klenow) fragment (from NEB) to blunt end the insert. or alternatively an EcoRV site can be designed into the primers so long as it is not present elsewhere in the insert. $\endgroup$ – Bez Oct 22 '14 at 9:43

To know if the ends are compatible see compatible cohesive ends (isoschizomers) from NEB website.

If you are unsure then do a blunt end ligation. For blunting a sticky end (From the comments):

Use Klenow or Pfu polymerase (or any proofreading/high fidelity polymerase).

You can use Taq if you intend to to a TA cloning.


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