2 replies to this topic

[jpopesku]

Hey all,
I'm at a crossroad and am seeking advice.
I have 2 dozen or so different PCR products being stored at -20ºC that I'd like to clone. The PCRs were performed with Invitrogen's PCR Supermix, which leaves a 3' A overhang.
I've read in various places that PCR products that are frozen "lose" their 3' overhang. In our lab, I have a choice of using a blunt-end vector, or one with the corresponding T' overhang.

So: Would you recommend using the blunt-end vector (with DNA ligase and all that)?
Or
Would you recommend re-adding the 3' A overhang with some excess Taq and using the overhang vector?

Thanks in advance for your input!

Edited by jpopesku, 17 March 2010 - 03:49 PM.

[pcrman]

I will try to avoid blut cloning. It is OK to store PCR products for a few days before ligation for TA cloning. Adding a bit polymerase an cycling a few times will work for sure.

[perneseblue]

I agree with Taqman. Avoid blunt end cloning. It can be more trouble than it is worth. Readding the missing A with treatment of taq and dATP for 20 mins, is a better path forward.