Cohesive end filling after CIAP treatment - (Feb/23/2011 )

Hi! I am planning a cloning strategy and I would like to know if a DNA polymerase (Klenow fragment or T4 DNA polymerase, for example) can work to make a cohesive end blunt AFTER the end was treated with alkaline phosphatase. In other words, does the polymerase need the 3' phosphate of a DNA strand to add new nucleotides to the strand?

Thank you very much for your help!

nephron

The phosphate on a DNA strand is at the 5' end. Why do you want to dephosphorylate before blunting?

Polymerase can extend a recessed 3' end after dephosphorylating and the resulting blunt end is still dephosphorylated I think.

Thanks for the response. I ended up changing my cloning strategy. Thanks again!

loucash on Thu Feb 24 09:54:09 2011 said: