niflab at biology.ucsc.edu
Thu Sep 16 21:01:31 EST 1993
>I have recently attempted to size the genome of an unknown gram positive
>bacterial species using inversion field agarose gel electrophoresis (using
>a Hoeffer controll unit) .
>I have two questions relating to this technique
>1. While I've been able to get good separation of bands from the yeast
>chromosome MW marker ( Boeringer manneheim's) I have found it impossible to
>get the largest chromosome (-+2.2mb) to migrate into the gel. Also ther
>appear to be two extra, fairly small bands (less than 10kb in size),which
>ar not mentioned in the catalogue. I was wondering whether, firstly, anybody
>knowsof a way of getting every band into the gel (there should be 17) and,
>secondly, what the extra two low mw bands are.
If you are seeing 16 bands on your FIGE gel, you are doing great. Two
of them in the middle co-migrate. A few years ago Phil Heiter made a
strain with one of them cut in half (integrated a centromere and telomeres).
Is the small band mitochondrial DNA? Circles would run faster than
their MW would lead you to expect.
>2.When undigested bactreial dna is run on the gel a badly defined band (
>a hazy smear really) is observed in the size range between 100 and 300 kb.
>Following restriction digestion this band dissapears so I presume it is DNA.
>Regardless of how gentle I am in preparing the DNA I can't get rid of it.I
>would like to know whether anybody has an explanation for this band's
>I would like to thank anybody with suggestions in advance
Is this the only band?
Robert Kuhn, PhD
University of California
Santa Cruz, CA 95064 USA
email: rkuhn at orchid.ucsc.edu
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