Blunt end ligation debate.

BENNY SHOMER 9238 pc386 at ccsg.tau.ac.il
Wed Jul 14 03:54:21 EST 1993


Hello Bionetters.

I would like to ask for people's opinions regarding the following issue:

I have performed a rather straightforward Blunt end ligation of a plasmid
vector, with a 208bp fragment of interest. The reaction was performed 
adhering the recommendations given is Sambrook et al Handbook ('Maniatis' 2nd
edition). i.e.- I used 1< j:i<3 ratio for the vector and 2:1 molar ratio of 
5' ends I:V. My vector was dephosphatized using CIP in standard protocol.
The ligation was performed with 15% PEG8000.

Following transformation, it was clearly evident that the CIP reaction failed
to perform, as I obtained many colonies in the control plate of Blunted/CIPped
self-ligated vector.

Now my debate: Should I "go for it" anyway, and try to pick up positive coloniesanyway, regardless of the fact that the CIP reaction failed, or should I insist
on performing a more efficient CIP, and discard the colonies I already have.

I don't mean to turn this into a tutorial of 'How to do ligations', as I believethis subject has been thoroughly covered by the group lately.
What I am interested in, is people's opinion on my question, and even better, if anyone had experience with such a situation (I believe many deed...), and what
were the results of the actions that were undertaken.

Thanks very much in advance. Benny.

 **************************************************************
* Benny Shomer                                                 *
* Tel-Aviv University                                          *
* Sackler School of Medicine, Dept.of Embryology and Teratology*
*--------------------------------------------------------------*
* Snail:  Ramat-Aviv , Tel-Aviv  69978 ,  Israel.              *
* E-mail :  pc386 at ccsg.tau.ac.il                               *
* Tel :  972-3-640-9238     FAX :   972-3-642-2046             *
*                                                              *
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