Does CIAP cause ligation problems?

jstrassw at OPAL.TUFTS.EDU jstrassw at OPAL.TUFTS.EDU
Fri Jan 10 13:38:00 EST 1997


Hello!
Boy, CIAP is a pain.  Do yourself a favor: buy Shrimp alkaline phosphatase
(USB sells it).  My standard protocol:
1. digest in whatever buffer
optional precipitate/resuspend in  the supplied SAP buffer)
2.add one microlitre SAP incubate 37 degrees 30 mijnutes
3. heat-kill 65 degrees 15 minutes 
4 load on your agareos gel and continue your cloning!
It really is a great product.
John


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John Strasswimmer, MD,PhD Candidate    | Phone (617) 636 8396
Tufts - New England Medical Center     | Fax   (617) 636 6190
Box 166                                | Email:  jstrassw at opal.tufts.edu
Boston, MA 02111 USA                   |                              
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On 10 Jan 1997, Robert Nicholson wrote:

> Guido Hooiveld wrote:
> 
> >For the last two months I am trying to perform a non-directed ligation.
> >Therefore I tread my vector with CIAP (Promega). However, till now I wasn't
> >successful.
> 
> 
> We have an axtremely good success rate with subcloning (blunt, sticky) into
> many different vectors, but the protocol we use is possibly a bit of
> overkill.  It is not time consuming and works, so we stick with it:
> 
> -cut vector DNA, phenol:chloroform extract, and ethanol precipitate.
> -wash ethanol precipitate with 70% ethanol and resuspend in 180 ul 10 mM
> Tris7.9.
> -add 20 ul of 10x CIAP buffer and 1-4 units of CIAP.
> -incubate 30 min at 37oC.
> -(often we add another unit or two of CIAP and incubate another 30 minutes).
> -add 3 ul of 500 mM EDTA8.0, 15 ul of 10% SDS, 1.5 ul of 10 mg/ml
> proteinase K, and 80.5 ul of water.
> -incubate 30 minutes at 56oC.
> -phenol extract once, phenol:chloroform extract once, EtOH precipitate
> (NaOAc), and 70% EtOH wash.
> -we then resuspend the vector and run it on an agarose gel and purify the
> linearized band.
> 
> 
> This is perhaps a bit anal, but it provides extremely clean and efficient
> vector.
> 
> 
> Robert Nicholson
> Resident, Department of Pathology
> McGill University
> Montreal, Canada (still)
> 
> 
> 
> 
> 




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