alkaline phosphatase...

AvV avvliet at ision.nl
Wed Aug 15 03:27:34 EST 2001


Hi Wolfgang,

I first digest, then add the SAP to digest, inc 15-30 min, heat kill and gel
purify. I don't like including it in the digest, since it increases chance
of nuclease introduction. Just my two cents..

hope this helps,
Arnoud


""Wolfgang Schechinger"" <wrote in message
news:3B7849F4.4158.7D1C5F at localhost...
> Hi all,
>
> Here is a debate going on if it is allowed (seems to some sort
> of dogma) to add SAP (or CIP) directly to a RE digest of a
> vector or better phosphatasing it in a separate step after
> purification of the digest. My opinion (and as I do it) is just add
> all to the mix and go, but the reason for the debate is how to
> minimize vector background when constructing a library.
>
> (digest is NgoMIV + XhoI to cut vector, BamHI to kill old insert
> in NEB Buffer IV and SAP)
>
> Wo
> [tiss mezzage wahs broduceRd using TYPO GENERATOR zoffwer]
> -----
> Dr. Wolfgang Schechinger
> Lab N233 (c/o Dr. Steve Roffler)
> Institute of Biomedical Sciences
> Academia Sinica
> 128 Yen-Chio Yuan Rd. Sec.2
> Taipei 115
> Taiwan R.O.C.
> Tel +886-2-2789-9152
> Fax +886-2-2782-9142
> Mobile +886-925-136893
> e mail wolfsc at ibms dot sinica dot edu dot tw
> -----
>
> ---





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