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pulse field >8 Mb fragments

Sylva Donaldson sylva at scar.utoronto.ca
Wed Mar 15 11:10:44 EST 2000


I've been trying to resolve >8 Mb fragments of restriction digested plant
chromosomes on pulse-field gels with no success.  I've been using a 0.8%
gel (chromosomal grade agarose), and a CHEF III apparatus, using the
optimum protocol for S. pombe chromosomes.  I've been struggling with
inconsistent DNA yield, and whether or not I digest the DNA, I get a smear
at 3 Mb, with no hint of higher molecular weight DNA on the gel.  I was
wondering if anyone has any experience with this.  I've toyed with the
idea that maybe the DNA is shearing as the gel runs, due to the force of
electorphoresis.  Other than that, I'm stumped.  I would appreciate any
help.  Please email me directly:  sylva at scar.utoronto.ca

Sylva Donaldson


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