Cutting phage DNA

David L. Haviland dhavilan at IMM2.IMM.UTH.TMC.EDU
Tue Nov 30 18:34:58 EST 1999


vivanova at my-deja.com wrote:
> 
> I am screening a gemomic library, and have identified several promising
> bacteriophage clones.  I am able to isolate a lot of phage DNA, but I am
> unable to cut the DNA with restriction enzymes.  Has anyone EVER
> experienced anything like this?  I have tried CsCl gradients and
> dialysing, but there is a glob of white stuff that is following the DNA.
>  It has been suggested to me that it is agarose.  If you think this is
> the problem, what brand of agarose do you use to plate the bacteriophage
> clones.  IF you have any other ideas as to what the problem might be, I
> would appreciate any suggestions.

Agarose is certainly a possibility.  But more so, are you using PEG
anywhere in the protocol?  If so, do no less than three 70% EtOH
washes.  PEG will inhibit all enzymes except for T4 ligase.

David
-- 
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David L. Haviland, Ph.D., Asst. Prof. Immunology 
University of Texas - Houston, H.S.C.
Institute of Molecular Medicine, R907
2121 W. Holcombe Blvd.,  Houston, TX  77030 
713.500.2413-Voice//713.500.2424-FAX
-----------------
If everything seems to be going so well, you have obviously 
overlooked something.
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