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 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
If everything seems to be going so well, you have obviously
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