Does CIAP cause ligation problems?

Guido Hooiveld g.j.e.j.hooiveld at farm.rug.nl
Thu Jan 9 12:29:01 EST 1997


Hi everybody,

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.

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!

Please post any suggestions here or mail directly to:
g.j.e.j.hooiveld at farm.rug.nl

Thanks in advance,

Guido
-- 
***************************************************************************
* Guido Hooiveld                              |                           *
* Groningen University Center for Pharmacy    |phone: (+)31 50 3637565    *
* dept. of Pharmacokinetics and Drug Delivery |       (+)31 50 3633272    *
* A. Deusinglaan 1                            |fax:   (+)31 50 3633247    *
* 9713 AV Groningen                           |                           *
* The Netherlands                             |                           *   
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