cloning of long PCR products

Tom Vink tvink at xs4all.nl
Wed Jul 24 02:36:09 EST 2002


Any repeat sequences in yoiur fragment ? Try stbl-2 e.coli's from
invitrogen. These are the only bugs I can propagate a special vector
without recombination.

grtz Tom


On 23 Jul 2002 18:03:33 +0100, subhi at gene.ccmbindia.org (Subhashini
Sadasivam) wrote:

>I amplified a 5kb region from human genomic DNA using Expand High fidelity
>system (roche). 
>After gel eluting the fragment , i tried ligating into pMOSBlue (
>Amersham) blunt ended vector. 
>I get white ("positive")colonies but on screening i find that a few
>contain insert...but of only 500 bp ...This 500 bp is at one end of the
>5Kb fragment i'm trying to clone...
>Does someday have an idea what's happening....
>i've tried it quite a few times ...with the same result...
>Please read and reply....
>thanks 
>subhashini.
>
>-------------------------------------------------------------------------------
>Subhashini Sadasivam,
>Ph.D student,
>Centre for Cellular and Molecular Biology,
>Uppal Road, Hyderabad - 500 007.
>India.
>Phone no: (040) 7192648 
>
>
>---




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