cloning of long PCR products

Subhashini Sadasivam subhi at
Tue Jul 23 12:03:33 EST 2002

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....

Subhashini Sadasivam,
Ph.D student,
Centre for Cellular and Molecular Biology,
Uppal Road, Hyderabad - 500 007.
Phone no: (040) 7192648 


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