IFAGE for sizing bacterial genomes

Darren Martin Martin at gene.unp.ac.za
Wed Sep 15 05:51:56 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.

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



More information about the Methods mailing list