get rid of Alkaline phosphatase

Dag Rune dag.rune.gjellesvik at biotec.as
Fri Sep 24 08:22:00 EST 1999


You can add SAP directly to the digest, too, but even simpler, you may add
the restriction enzyme AND SAP to the vector (simultaneous digest and
dephos). The vector is dephosphorylated as soon as it is cut.

Just be sure to calculate the correct amount of SAP for the incubation time
for the restriction digest. (Units = 1 / [#hours of incubation]).

If your restriction enzyme is heat-labile (and your'e using SAP), then kill
BOTH enzymes by heating.

I've tested this method on direct religation, and it worked well, especially
for 5'-protruding and blunt ends. For 5'-recessed ends, you may use a little
more SAP if you get too high background, but it should work OK with the
recommended amount.

Dag Rune Gjellesvik
Biotec ASA, Tromso, Norway

Jill Johnston wrote <7r6njl$m1e at ds2.acs.ucalgary.ca>...
>I routinely use QiaexII from Qiagen to clean up after CIAP.  I get very low
>religation.  I also follow the procedure that appeared in BRL's bulletin a
few
>months ago in which you simply add the CIAP directly to the digest.  Works
>great!
>
>
>Sue Jin wrote:
>
>> Hi, there
>>
>> I am wondering  if  there is a easy way to get rid of the posphatase
>> without doing phenol extraction after dephosphorylation of the plasmid
DNA
>> for subcloning? Your help is appreciated!!
>>
>> sue
>
>--
>Jillian Johnston
>Dept. of Biochemistry and Molecular Biology
>University of Calgary
>Phone (403) 220-3559
>Fax     (403) 270-0737
>
>





More information about the Methods mailing list