Cloning problem

John Watson watson_j at bms.com
Tue Feb 11 16:13:07 EST 1997


Brendan Scott wrote:
> 
> We have been having an extremely dogged cloning problem.  We take microbial
> genomic DNA, treated with CsCl, and do a partial digest with Sau3A.  We
> ligate it into pUC 18 and transform it, and it works fine.  BUT we are
> trying to make a library of fragments larger than 2kb, so we have tried
> fractionating the partial digest with several different fractionation
> methods, and then ligating, etc.  Regardless of fractionation method, we
> can't get the cloning to work!
> 
> Does anyone have any suggestions?

People in the lab where I postdoc'd used to make BAC libraries out of 
100-250 Kb DNA.  Their preferred method was to partial digest, gel 
electrophorese, isolate the region of interest, gelase treat, and clone.  
Seemed to work OK for them.


AJW
----------------
John Watson
Bristol-Myers Squibb Co.
watson_j at bms.com
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