If you trying to say that I could just add a proofreading polymerase to a finished PCR reaction that may be a bit stupid, but I wondered if someone has a protocol, how much enzyme for how long, same as the Taq treatment. Sure someone has it if it's that simple.

In any case what I meant is how to do it when I already HAVE my PCR product done with Taq polymerase that already makes A overhangs and THEN I want to blunt it. And for any reason I don't want to do the PCR with a proofreading polymerase instead.

you can use klenow fragment or t4 dna polymerase.

Trof on Mar 4 2009, 06:03 AM said:

Actually, you don,t need to do anything as Taq only add 3'A to small percentages of its products.

Hi all,

Iam new to this topic.
is it that only for blunt end cloning ,we have to dephosphorylate the vector . So, CIP treatment is not necessary for ligation of cohesive ends of vector and insert.

Thanx

novagen on Feb 7 2010, 09:08 AM said:

yeah, u r right. for cohesive ends, u do not require to give CIP treatment. In blunt-end it becomes necessary, to avoid self-ligation.

popogirlxd on Jan 3 2005, 10:09 AM said:

I think its because repeated freeze thaw degrades ATP.

Yes, SAP works very well in my experience. Definitely should dephosphrylate the vector before ligation.

Research Papers

hi everyone..just drop by to ask a question regarding the ligase buffer. I know there are salts in the buffer (ready made buffer from company) but is it normal if we can see some precipitation in the buffer? it looks like salt precipitation. And my lab colleague didn't remember when they bought this ligase buffer.

William Parkar on Thu Apr 22 06:16:17 2010 said:

Warm it in your hand, maybe vortext it a little.
This happens frequently with NEB T4 Ligase buffer (10x) and it goes right back into solution.