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ww40 William_D_WARREN at UMAIL.UMD.EDU
Fri Jun 18 09:59:00 EST 1993

Carol Enderlin wrote:

>What's the largest (and smallest for that matter) fragment that
>bionetters have purified by geneclean and successfully cloned.
>I'm asking because I wanted (note past tense suggesting less than
>success in first tries) to subclone large fragments from cosmid
>clones.  My two fragments are slightly smaller and slightly larger
>than 20 kb, respectively. One came out as a complete smear, the other
>had less smear, more of a band, but didn't look quite as large as
>the starting band in the digest.


Although I have no first hand experience with this a graduate student in a
previous lab that I was in used geneclean to gel purify the 15-20 kb region
of a partial sau3A diest for library construction quite sucessfully. The
problem with gencleaning large fragments is the potential for a single
molecule to become attached to more that one glass particle. Thus vigorous
resuspension of the glass milk during washing will cause strand breakage and
fragment size degradation. To minimise this do not resuspend your geneclean
pellet in the wash solution, but simply rinse it is as you would ethanol
rinse a DNA pellet. Since this is clearly a less thorough wash that the
recommended resuspension, it needs to be done several more times than you
ordinarily would wash the glass milk pellet. Just let
the wash solution sit in the tube for several minutes during each step,
flick the tube one or twice and recentrifuge briefly before removing the wash
each time. THen when it comes time to resuspend the DNA do this gently after
letting the pellet soak for several minutes.

Hope this helps


William D. WARREN, PhD
Center for Agricultural Biotechnology     Email: ww40 at umail.umd.edu
University of Maryland at College Park    Phone: (301) 405-7681 


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