Which should be CIPed, 7K one or 3K one?

Hiranya Roychowdhury hroychow at NMSU.EDU
Wed Aug 26 10:00:53 EST 1998


At 08:50 PM 8/25/98 -0700, shengyang at usa.net wrote:
>Hi, all!
>
>What you thought is not the point of the question. Because, the two
>fragments are both cut out by ~ScaI~. You know, ScaI cut ampR into 2 parts.
>So, the 3k one carries one part of ampR and the 7k one carries another part
>of it. Then which one is the vector and which one is the insert? I think it
>is hard to say.
>
>One of the fragments contains the ori, but neither contains the ampR. If
>re-circularized and up-taked by the competent cells, one can not replicate
>and the other can but its host can not grow on LB-Amp plate. So, I consider
>that the background is rather low. The transformants which appear on the
>plate should contain recombinant plasmid very likely.
>
>The reason why did I consider that one of the fragments should be CIPed is
>that I want to increasing the effiency of the ligation. 3k + 7k => 10k. It
>is said that the reaction is difficult. The product is too large, more, the
>fragments are blunt-ended and it is easier for they to re-circularized.
>Though the re-circularized plasmids can not survive at last, they competent
>the competent cells ...
>
>Thanks for your advice again!
>
>Yang Sheng

The scenario you describe does not need removal of PO4.  Since the ori and
the ampR have been separated, there is little chance of recovering anything
else. Concatenation may be expected, but the desired plasmid will
predominate. We routinely ligate and transform with plasmids >10kb, so it is
not quite as unthinkable as people tend to believe. Hence, you should be
able to obtain a large enough # of transformants to screen for the right
plasmids. 


Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu




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