HELP! - cloning problem

Daniel Schlieper aeg17 at campfire.rrz.uni-koeln.de
Wed Nov 4 10:56:21 EST 1998


Hello Mari,

sounds like you try to clone a toxic gene. Does it work to clone it
into a completely other vector? Like a non-gene region of pB322, or do
you need it in pBAD or pMAL?

Regards, Daniel

MSRA at novo.dk (MSRA) writes:

>      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.
>      
-- 
Daniel Schlieper                    Institut fuer Biochemie
                                    Zuelpicher Strasse 47
Daniel.Schlieper at Uni-Koeln.De       Universitaet zu Koeln 
Tel.: +49 221 470 6443              D-50674 Koeln, FRG




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