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

Patrick F.H. Lai pfhlai at LOOKSMART.COM
Wed Aug 26 00:46:41 EST 1998


---- Begin Original Message ----
>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
>_____________________________
>WO SHI NI DE BEN XIONG XIONG!
>
---- End Original Message ----

Well, in case you do not see any colonies at all, i would suggest 
removing all of the vector sequences first.
Hope you can find 2 useful non-blunt restriction sites at the MCS.
A 3-fragment ligation should not be too difficult.

Pls ignore my other response.

BTW, what is a 'ben xiong xiong' ?
I apologise for my broken PuTongHua.
Xie xie.   ;-)






Hope this is useful info.   :-)


Patrick F.H. Lai  < PFHLai at looksmart.com >
Graduate Student
University of Toronto
Toronto, Ontario, Canada





       
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