What's wrong with my cloning?---Help!

Martin Kennedy mkennedy at chmeds.ac.nz
Mon Nov 22 00:39:07 EST 1993


In article <01H5JZ6BH4EQ9ODRJ5 at UCBEH.SAN.UC.EDU>, WANGNN at UCBEH.SAN.UC.EDU writes:
> I am sub-cloning my c-DNA (1.5kb) from pGEM2 to pBluescript SK+ because I need
> the restriction sites and the orientation for further studying.  The vector
> (pBlue.) is digested with EcoR I and treated with CIAP.  The cDNA fragment is
> the EcoR I fragment cloned in pGEM2 purified from the gel (and I also try the  
> other methods to get this fragment).  Then the ligation has been finished with
> GIBIO T4 ligase.  The transfected E.coli. (XL-1) is selected on the
> Amph./Tetracyclin with X-gal/IPTG.  The results on the plates is beatiful: 
> Blue is blue, and white is white; low percentage of blue clonies.  After each
> clone is cultured in 5ml LB with Amph./Tetra., the plasmid DNA is puified using
> the Promega Magic-miniprep kit.  However, I am surprised that EcoR I digestion
> only offer a 4.5kb fragment (linear) on the gel.  It seems that one EcoRI site
> is lost.  I have tried every kinds of EcoRI and and other purification method
> of plasmid.  Untill now, there at least 4 different ligation have been done and
> at least 50 bacterial clonies have been checked.  The seem result is offered: 
> EcoR I can only give a 4.5 kb fragment, and no 1.5kb of insert at EcoR I site
> has been shown on the gel. 
> 
> Hong F Wang 

I'd put my money on a contaminating plasmid present in either your
electrophoresis buffer, your ethanol, or any other reagent.  Try self-ligating
your insert without any vector present, and see if you get any whites. This
sort of problem is more likely than spurious and consistent loss of one R1 site
during cloning (though it can happen). 

The best way of finding troublesome clones in amongst a white background is 
to use colony lifts, and hybridize with some of the labelled cDNA you are
trying to clone.  I'd give up making minipreps 'til you find a positive clone
by hybridization.

-- 
Cheers,

Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
NN  NN NN           Christchurch School of Medicine            ZZZ
NN   NNNN              Christchurch, New Zealand              ZZZZZZZ
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