Does CIAP cause ligation problems?
ladasky at leland.Stanford.EDU
Thu Jan 9 18:19:19 EST 1997
In article <5b39st$6qv at info.service.rug.nl>,
Guido Hooiveld <g.j.e.j.hooiveld at farm.rug.nl> 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
>After transformation the ligation mixture in JM109 bacteria I obtained several
>(app. 100) colonies which were AMP resistant. But all these colonies didn't
>contain the right construct as shown by restriction analysis, however, they
>all contained the same, non-cleavable (?) plasmid as seen by electrophoresis.
>This happened already twice. I select bacteria with AMP-75.
>I was told the use of CIAP to dephosphorylate the DNA fragments may likely be
>the cause of my ligation problems.
>So my question is: is this correct; have other people experienced the same,
>and if so, how did you solve this; what is the best way to get rid of the
>CIAP? Other suggestions regarding this problem are also most welcome!
I have been going through something very similar. I have performed
many ligations, both directional and non-directional, and I always used CIAP.
The same protocol has always worked in my hands with any vector and template
that I have tried. Suddenly, I came across this ligation that simply would
not work. I would plate out my entire transformed cell mixture and obtain
only a dozen colonies or so, and none of them would contain inserts.
By reading through various references I've discovered that I have a
lazy-man's protocol that doesn't make much of an effort to remove residual
enzymes, either the restriction enzymes or the CIAP. My latest round of
cloning has given me many more colonies. In order to increase my yields,
I have had to exclude CIAP altogether and rigorously inactivate my restric-
tion enzymes before proceeding to the ligation step. The way that this is
done is to add EDTA to a final concentration of 15 mM, incubate 15 minutes
at 65 degrees, than extract twice with 1:1 phenol/chloroform. If you must
use CIAP, you can incativate it using this same approach but the sample
must be heated to 75 degrees!
I'm not sure whether the problem is my insert or my reagents. I
used up a vial of CIAP a little while ago and bought another one from a
different source. The concern about excluding CIAP from the ligation is
that, if you accidentally dephosphorylate your insert, it won't ligate.
But I never had this problem before... do different sources of CIAP vary?
Has this enzyme been cloned or is it still isolated from calf intestine?
Is CIAP the actual culprit, or are there other nucleases present in the
CIAP preps that do the collateral damage?
Unique ID : Ladasky, John Joseph Jr.
Title : BA Biochemistry, U.C. Berkeley, 1989 (Ph.D. perhaps 1998???)
Location : Stanford University, Dept. of Structural Biology, Fairchild D-105
Keywords : immunology, music, running, Green
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