Sticky ends to blunt - (Aug/15/2005 )

Hi again

Right. This will be a really obvious question but I cant remember what the answer is......

Whats the best way to convert sticky ended restriction digest products to blunt ended?? I think its something to do with Kelnow polymerase but i dont really undestand the protocol. Has anyone got a decent protocol or an alternative method???

Cheers

P

Hi, I use NEB DNA Polymerase I, Large (Klenow) Fragment, along with dNTPs, and appropriate buffer at RT for 60' which works fine. This can be used for blunting ends by 3' overhang removal and 3' recessed end fill-in.

Can you PCR with a proofreading enzyme?

I like to use T4 DNA polymerase. (NEB)
It is good for 3´ overhang removal to form blunt ends and 5´overhang fill-in to form blunt ends.

There are two ways to get the blunt-end.
I usually use proofreading enzyme if I would like to have a blunt-end cloning.
The other way is to use DNA Polymerase I, Large (Klenow) Fragment. It's a bit trouble because one more step is needed.