3' overhang dephosphorylation? - dephosphorylation problems after KpnI dig (Aug/26/2004 )
Hi, I`m having trouble in dephosphorylating my vector after KpnI cutting ( 3` overhang), I tried using BAP at 60C for 1hr but still I have a lot of colonies in the negative control plate (just vector). Does anyone have some advice? Please help me!!!!!
did you use proper buffer,when you dephorsphorylate?
Yes, I did. And I used large excess of enzyme respect to pmol of DNA end.
Did you check the quality of restriction digest?
maybe there's some plasmid left uncut? Have you tried transforming your "cut" vector without ligation?
I use SAP (shrimp alkaline phosphatase) at 37°C for 1 hour, and that works everytime...