tfitzwater at somalogic.com
Tue Aug 14 09:24:37 EST 2001
>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)
I have routinely included SAP in my double digests of vectors for
several years on the premise that elimination of an extra cleaning
step between the two procedures cuts down on any loss of DNA.
80 Units each of BamH I and Hind III and 3 Units of SAP are
used to digest 25 ug of pUC9 in a 200 uL reaction in 1x KGB
for 3 - 3 1/2 hours at 37 degrees.
(SAP: 14.425 pmols pUC9/25 µg plasmid DNA x 2 ends =
28.85 pmoles of 5' ends x 0.1 unit SAP/pmol = 2.9 units SAP
/25 µg linearized pUC9.) I use 0.5 pmoles of vector per
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