HELP! - cloning problem

MSRA MSRA at novo.dk
Wed Nov 4 02:25:36 EST 1998


     Hello,
     
     We have a strange cloning problem (and totally frustrated!).  After 
     the blue/white selection, we tried insert check by colony PCR.  Here, 
     most of the case, there is no band amplified, even though we selected 
     white colonies.  We also tried minipreping plasmids and checked with 
     restriction enzyme cuts.  Plasmids are there, but we get different 
     sizes of digested fragments - total size of appeared fragments are 
     shorter than what we expect.
     
     Our vector is 5-6 kb (pBAD and pMAL) and the insert is 2 kb.  Yes, I 
     have to add that this problem started ever since we tried to clone in 
     the 2 kb insert to these different vectors.  We tried two kinds of  
     cells, DH12S and JM109, by electroporation and heat shock, 
     respectively.  But both showed similar results - while colonies, 
     plasmids are there, but wrong ones.
     
     I wonder whether some kind of plasmid recombination happened here...?  
     However, the DH12S cell is recombinant deficient strain (recA1-).    
     Does anyone out there know whether it's worth trying other cells such 
     as SURE?
     
     Any comments and suggestions are greatly appreciated.  Please help us!
     
     Yours,
     Mari
     



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