dephosphorylation by SAP

killer yeast vvsvetlov at utmem1.utmem.edu
Mon Jul 15 19:46:30 EST 1996


In article <Pine.SUN.3.91.960711132751.17038B-100000 at verdi>,
hroychow at NMSU.EDU ("H. ROYCHOWDHURY") wrote:

> Hello All,
>         I had been using SAP lately. I use it the same way as I would CIP,

We don't use SAP buffer adding enzyme instead to normal digest buffers -
so far we tested Boehr. H & B, Universal from Stratagene and 1forAll from
Pharmacia. It appears that extending incubation of 1u per 5mcg of 5-10 kb
plasmid to 3 hours effectively cuts background to below 5%. Recently I
started adding SAP to the digest from the beginning - including o/night -
with background falling to the 0 w/out visible drawbacks. I kill SAP for
30 min at 70C and use the resulting solution in cloning directly or spin
it through Chromaspin -100, never PCI unless needed to get rid of other
enzymes (BamHI). Degree of dephosporylation as judged by removal of
radiolabel is in the 95-100% and in all investigated cases when people
have troubles with it in the lab they were using some peculiar digest
buffer (SAP does have some salt requirements) or the plasmids were not
fully cut to start with. See if longer incubation does the trick for you.
Regards,
Vlad

-- 
Of what use is a philosopher who does not hurt anybody's feelings?
                                Diogenes



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