HELP! - cloning problem

Mr K Dai kdai at hgmp.mrc.ac.uk
Fri Nov 6 12:45:08 EST 1998


>      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 don't think there is problem with recombinant. First I want to know that
if you are cloning the PCR fragments ? Make sure that your PCR products is
the right length and right amplification (do restriction mapping if you
have some information of the interested region, or nested PCR). Second,
before you transform your constructs, please check your host. It will be a
simple test: grow your host cells with or without antibiotics to see if
there is any existing plasmids in your host, and include negative control
whichever tranforming method you are following. 

Hope this will help!

David Dai




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