FIGE

[Ludwig Lab-UCSC]

>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 
>occurence. 
>
>I would like to thank anybody with suggestions in advance
>
>Darren Martin

Is this the only band?