Gel Purification -- Anyone else have this problem?
goldberg at bms.com
Mon May 19 07:28:36 EST 1997
In article <337FBECD.18F0 at ukcc.uky.edu>, walush0 at ukcc.uky.edu wrote:
> Hello molbio experts,
> Our lab has had a reoccuring problem (nightmare) with gel purification
> and subsequent ligation of fragments using a couple of commercially
> available kits (Geneclean and Qiaex gel extraction kit).
> Any thoughts or suggestions are most welcome.
> Thanks, Warren..
If you were unable to recover your fragment or if they would not ligate
at all, then I would be suspicious of the kit, but you said that the
yields were fine and you were able to obtain clones after ligation and
transformation. So I would look into your host strain and/or insert
itself as the source of the problem. I have had instances were fragments
mysteriously rearrange or delete DNA...my assumption in such cases is that
the host cells doesn't like the structure of the DNA or the product being
expressed and selects for a more palatable fragment. You might try using
different host strains and see if one of them can tolerate your inserts
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