pulse field >8 Mb fragments

Choona xuna68 at hotmail.com
Thu Mar 16 12:47:11 EST 2000


"Sylva Donaldson" <sylva at scar.utoronto.ca> wrote in message
news:Pine.GSO.4.10.10003151109540.2003-100000 at banks.scar...
> Greetings,
>
> 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


Hi

Some one in my lab said look at :

Genomics 52, p1-8, 1998.
Osoegawa et al An Improved approach for construction of BAC libraries.

They don't go as big as 8 megs but it may help.

NB They use a CHEF kit too.

HTH

Choona






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