Sma I religation problem
Mad Dan Eccles
rpgrant at molbiol.ox.ac.uk
Wed Jul 26 11:16:03 EST 1995
In article <BARRYM.95Jul20125808 at stella.med.utah.edu>, barrym at stella.med.utah.edu (Barry Moore) writes:
>> Should still work, but you need to phosphatase the vector.
> I would add that you want to be careful with the CIP. I just spent the last month figuring out that
> I was using to much CIP, and it was ruining my cloning (I don't know if this enzyme is so harsh that
> it was chewing up my vector ends, or if it was making it through my gel purification to destroy my ligation).
> I found that 0.1 U/0.5-1.0 ug vector prevent recircularization.
I am _very_ careful with CIP; I never use it. (!)
With the usual disclaimers, I find that Shrimp (yes, I said Shrimp) AP from
USB/Amersham is a beaut of an enzyme, and we now use it routinely, even for
blunt ends. Never had experience with -OH overhangs, but needles to say it's
grreat with -P overhangs.
Richard P. Grant MA DPhil rpgrant at molbiol.ox.ac.uk
Nuffield Department of Obstetrics and Gynaecology, University of Oxford.
Call yourself a dog? I've seen better hairs on a lavatory brush!
More information about the Methods