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

WANGNN at UCBEH.SAN.UC.EDU WANGNN at UCBEH.SAN.UC.EDU
Sat Nov 20 22:51:19 EST 1993


Hi netters:

I am posting this message for any possible answer.  My question is very simple. 
But I can not find the answer.

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. 

Can some one tell me what is going on about this EcoRI cloning?  Is one of
EcoRI site a hot spot of mutation?  Or is there any problem in this cloning?

Any suggestion will be appreciated !

Best Regards.

Hong F Wang 
Dept. Anatomy and Cell Biolgy 
Univ. of Cincinnati
231 Bethesda Ave.
Cincinnati, OH 45267
Phone: (513)558-6787
Fax: (513)558-4454  



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