get rid of Alkaline phosphatase

Keith Rand Keith.Rand at molsci.csiro.au
Fri Sep 10 02:06:49 EST 1999


In article <4.1.19990908145127.0092eec0 at sjin.pobox.stanford.edu>,
sjin at STANFORD.EDU (Sue Jin) wrote:

> Thank you for all the helps. The phosphatase I am using is SAP. However, I
> phoned the Roche company where we bought the enzyme from. They suggested me
> to do the phenol extraction for the ligation since ligation is picky
> sometimes. Does anybody have experience with SAP without doing phenol
> extraction and also have good result of ligation? thanks!!
> 
> sue

At least in my hands, it's not necessary to remove the shrimp alkaline
phosphatase. I use 2 or sometimes 4 uL of the phosphatased DNA in a 20uL
ligation. When I first bought the enzyme I did the simple test of plus and
minus phosphatase treatment and found that you get the same number of
clones for both treatments. The major difference is, of course, the near
total elimination of  any background. There was a thread a while ago about
trouble with this enzyme, so maybe some people have been unlucky and
received a bad batch.

Thanks to the people in this newsgroup who recommended this enzyme a few
years back because it certainly simplifies things!

-- 
Keith Rand,  Sydney Australia



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