CIP or BAP
Blum Beat
requesting information about the use of CIP (calf intestinal alk. phosphatase)
or BAP (bacterial alkaline phosphatase) in order to remove
the 3' terminal phosphate from an RNA oligo. Which of the two works better for
this special purpose?
All information concerning the topic is appreciated.
Thanks!
Carlisle Landel
The enzyme of choice CIP, because it can be heat inactivated by incubation
at 65 degrees, while BAP requires a phenol and chloroform extraction for
inactivation. CIP is *very* efficient in KGB (or "universal") buffer--simply
add a few units at the end of your digestion and incubate for 5 more minutes
at 37 degrees, then heat inactivate. Be careful not to incubat much longer,
since CIP does have a problem that it seems to chew up the ends of your
DNA if you let it go too long, making your fragment unclonable.
Carlisle
Bernard Murray
> In article <35bo43$2...@aragorn.unibe.ch>, <beb...@ubeclu.unibe.ch> wrote:
> >RNA editing research group in Bern is
>requesting information about the use of CIP (calf intestinal alk. phosphatase)
>or BAP (bacterial alkaline phosphatase) in order to remove
>the 3' terminal phosphate from an RNA oligo. Which of the two works better for
>this special purpose?
>
>
> at 65 degrees, while BAP requires a phenol and chloroform extraction for
> Carlisle
Another alternative is Shrimp Alkaline Phosphatase as this is very sensitive
to heat inactivation but is reportedly otherwise the same as CAP. I've used
SAP successfully but haven't performed a direct comparison. However, the
knowledge that the enzyme is efficiently killed is one less worry. I
don't have the deatils handy so I can't give you the supplier. I suspect
it is somewhat more expensive than CAP but one tube lasts for ages.
Bernard
Bernard Murray, Ph.D.
ber...@elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
Johnny D
Has anyone else out there tried SAP (Shrimp alkaline phosphatase Available
from USB). This is completely inactivated at 65 C for 15min without the
addition of any EDTA or anything else. This allows you to use your SAPed
vector immediately without having to extract/gel purify.
It seems to work OK in our lab but not as efficiently as CIP (Boehringer)
which isn't easy to get rid of. However I was CIPing and SAPing for 1
hour. Perhaps I was/am incubating them for too long (see above).
matthews
add 0.5U SAP per microgram of vector DNA directly to my restriction
digests, incubate for 1hr @ 37C and then heat to 68C for 30 minutes to
kill the SAP. At this stage I normally gel purify the vector before
ligation.
David Matthews
Ian A. York
religation after CIP. I n the least few weeks I've tried following the sug-
gestion posted here some time ago (essentially: read the instructions, dummy)
and used the recommended amount of CIP for my DNA (which involved diluting the
CIP about 1/100) - the results were beautiful on the couple of clonings I've
tried this way.
I don't gel-purify my DNA; I heat-killed the CIP (75oC, 15 minutes -
I think; whatever the NEB catalogue suggested) and then used GeneClean to
purify the DNA from solution. Worked fine.
I've used the GeneClean, by the way, for several clean-up-DNA pur-
poses - from PCR, from BAP and CIP, from T4 DNA pol - and it works well. I'm
sure many other people use this - it is one of the things geneClean is mar-
keted for, after all - but I haven't seen it explicitly mentioned here so I
thought I would do so.
Ian
Andy Law (Big Nose)
jp...@mole.bio.cam.ac.uk (Johnny D) wrote:
>
> Has anyone else out there tried SAP (Shrimp alkaline phosphatase Available
> from USB). This is completely inactivated at 65 C for 15min without the
> addition of any EDTA or anything else. This allows you to use your SAPed
> vector immediately without having to extract/gel purify.
>
> It seems to work OK in our lab but not as efficiently as CIP (Boehringer)
> which isn't easy to get rid of. However I was CIPing and SAPing for 1
> hour. Perhaps I was/am incubating them for too long (see above).
We used to use CIP, now we use SAP for the very reasons you describe. I
haven't ever done any real comparisons but I reckon you are probably right
but when you say that it isn't as efficient as CIP. However you are
definitely right about CIP being harder to get rid of and that's why we
have stuck with SAP.
Andy Law
( Lawa @ bbsrc.ac.uk Big Nose in Edinburgh )
Tim Buss
We also now use SAP and are perfectly happy with it. There is also a
product called HK phosphatase that I used a few years back. Like SAP it
too could be heat inactivated. Possibly marketed by Epicentre
Technologies??
Tim.