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
> Benjamin Braun
> bbraun at ucla.edu
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.
|\ | | / \ / | 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"
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