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

shengyang at USA.NET shengyang at USA.NET
Tue Aug 25 22:50:59 EST 1998


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
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WO SHI NI DE BEN XIONG XIONG!




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