DH10B/P3 strain of E. coli

Michael Benedik benedik at uh.edu
Thu Aug 7 13:43:43 EST 1997


In article <l03110705b0053ec23821@[128.143.75.64]>
mtw3p at AVERY.MED.VIRGINIA.EDU (Mark Worthington) writes:

> I am trying to find DH10B/P3 cells -- these were formerly available from
> GIBCO, but when they discontinued the selling product, they destroyed their
> stocks.  They suggested I try to find someone with a glycerol stock who
> would be willing to provide an aliquot.  Help!   It is not to be found in
> my searches of the ATCC or the E. coli Genetic Stock Center at Yale.
> 
> (I have MC1061/P3, but this is endA1+ and the DNA looks like it.  The
> library I am prepping is in a supF backbone, so I am locked into a strain
> containing the P3 episome).
> 
> Thanks in advance for your help.
> 
> --Mark
> 
> ------------------------------------------------------------------
> Mark T. Worthington, M.D.
> Division of Gastroenterology and Hepatology
> Department of Internal Medicine
> University of Virginia Health Sciences Center
> P.O. Box 10013
> Charlottesville, VA 22906-0013
> ------------------------------------------------------------------
> 
> Phone (804) 243-2652 /(804) 243-2655 Secretary
> 
> Fax (804) 243-6169
> ------------------------------------------------------------------
> Fedex address/office:  Room 1036, MR-4 Building, Lane Road
>                 University of Virginia Health Sciences Center
>                 Charlottesville, VA 22908
> ------------------------------------------------------------------
> 
> 

Three suggestions:

1) I often miniprep DNA from endA+ strains. The secrete is either to
phenol extract in order to inactivate the endonuclease, or else to not
treat with RNAse. the crude RNA is an inhibitor of the endA protein and
if left intact will serve to protect your DNA

2) transform the P3 plasmid into your favorite strain. Miniprep your
MC1061/P3
strain and retransform (preferably by electroporation because
tranformation with large DNA is very inefficient.) You only need 1.
After all P3 is a plasmid, just very very big.

3) transfer the P3 plasmid by mating. I can help if you don't know how
to do matings,but basically you need genetic marker to select for the
recipient strain such that the donor (MC1061) won't grow.

By the way, according to the Gibco/BRL catalog DH10B is also endA+, so
this won't really do you any good.

I also don't believe what they told you. They never would have
destroyed their stocks. Never. I would try to contact one of the
scienctists there instead of one of the customer service reps. for
example whoever was on the original P3 papers they published in Focus.


Michael Benedik
Department of Biochemical Sciences
University of Houston
benedik at uh.edu



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