4 replies to this topic

[giuxy]

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!!!!!
Thanks
Giusi

Edited by giuxy, 26 August 2004 - 05:33 PM.

[kroger]

did you use proper buffer,when you dephorsphorylate?

[giuxy]

Yes, I did. And I used large excess of enzyme respect to pmol of DNA end.

[jadefalcon]

Did you check the quality of restriction digest?

maybe there's some plasmid left uncut? Have you tried transforming your "cut" vector without ligation?

mike

Edited by jadefalcon, 14 September 2004 - 08:17 AM.

[Nina]

I use SAP (shrimp alkaline phosphatase) at 37°C for 1 hour, and that works everytime...

/Nina