Taq/PFU polymerase A overhang - bad for blunt ending? (Apr/12/2006 )

Hello-

I have been having a bit of trouble blunt end cloning using SmaI to digest my vector and PCR to amplify my insert. I had assumed (i don't clone too much) that the PCR product would be ok for insertion into a blunt ended SmaI site. MY Friend says it may not be working because Taq and/or PFU leave short overhangs.

IS this a problem for blunt ended cloning? Can I Use the Klenow fragment to cut off/fill in the overhang and then clone into my blunt ended SmaI site?


Thanks a lot,

Mountainman

Taq leaves an extra A but Pfu doesn't. Mixtures (blends) of taq and a proofreading enzyme will produce products that of which some contain the extra A (from taq) and others without it (removed due to proofreading activity).

If your PCR is done with Pfu (or any other pure proofreading enzyme) it should be fine for blunt ligation.

Why not make the primers with the SmaI site and cut?

The PCR product will have a OH instead of a 5' phosphate...are you using a kinase? You can't form a covalent bond without the phosphate.

-Matt

Thanks for the replies-

So if I understand correctly, even if I use a non-overhang creating enzyme, like pfu, I will still have to use a kinase to get ligation because of the missing phosphate in the PCR product?

I suppose that's a bit faster than redesigning primers with SmaI sites, which I was about to do....

Thanks,

Mountainman