Rapid Ligation

Nigel Kenward mbxnjk at vax.nott.ac.uk
Thu Feb 15 07:10:10 EST 1996


bbraun at ucla.edu wrote:

> Very interesting!
> 
> BTW, could you please post or e-mail your protocol for
> phospatasing the EcoRV-digested vector?  I have tried this
> but got poor results, presumably because CIAP is less active
> on blunt ends.  But you obviously achieved excellent
> phosphatase treatment.  This could make my life quite a bit
> easier.
> 
> TIA,
> Benjamin Braun
> bbraun at ucla.edu


Benjamin,
Again, there is no trick. Buy a good CIAP (we use promega), look after it, use their buffer 
and a tiny amount of the enzyme ( as suggested by most protocols ) and away you go.
To get good ligation it's worth phenol-chloroform after CIAP and a good quantitation of 
vector and insert.
I used to get poor results too, but if you take No shortcuts, it's easier now.
Good Luck,
Nigel
-- 
            ____     ___
|\   |  |  /    \   /     |         NIGEL KENWARD,
| \  |  | |   ___  |__    |         Dept. Biochemistry,
|  \ |  | |     |  |      |         Nottingham University Medical School.
|   \|  |  \____/   \___  |___      Clifton Boulevard.
-------------------------------     Nottingham. NG7 2UH.
 Tel (0115 924 9924) ext 44789      Fax (0115 942 2225)
  email:mbxnjk at vax.nott.ac.uk      "You can tell me.......I'm a doctor"



More information about the Methods mailing list