I need to dephosphorilate blunt ends and, surfing on internet I found this
two enzymes:
- Shrimp Alkaline Phosphatase (SAP)
- Calf Intestinal Alkaline Phosphatase (CIP)
Have you any experience about these phosphatases, I mean wich is the best
for blunt ends?
Either will do dephosphorylation part just fine. SAP is A LOT easier to
kill, so other things being equal, go with it. That said, more frequently
than not, using phosphatases creates more problems than it solves
(precisely because of the incomplete kill). If it is blunt ligation you are
thinking of, better way of doing is to use huge excess of insert
(say, >30-fold). Works fine.
Also worth mentioning: In one special case blunt ligation is a piece of
cake. If it is a single blunt site you are cloning into, chances are that
ligation of your insert will destroy this restriction site. If you are
lucky and your insert does not have the same site then do this: ligate in
the presence of large excess of insert, heat kill ligase, digest the
reaction with that blunt cutter, transform. Since linear plasmid
is >1,000-fold less transforming in E.coli, essentially none of your clones
will contain self-ligated backbone.
Dima
