purification of 140 Kbp from agarose

Ghis Allard ak302 at FreeNet.Carleton.CA
Tue Feb 20 08:58:24 EST 1996


Hi,
	lately I have been trying to isolate a pulse-field gel
fragment from agarose. GeneClean didn't work at all so I went on 
with an agarase digestion of the agarose followed by a dialysis 
over a millipore membrane sitting on a becher full of TE. This 
dialysis usually work very well for smaller fragment. But after
this step, I precipitate with 1/10 volume of 3M NaoAc and 3 volume
of cold 95% Ethanol, rinse with 80% ethanol and resuspend in 10 ul 
of dH2O, run on a gel and there is nothing. I even hybridised it to
see if there was a trace amount. But there was nothing. I also tried
a phenol extraction with no dialysis followed by the precipitation,
and still nothing. I also tried this thing called GeneCapsule, which
uses electrophoresis to get the DNA out of the agarose and onto a 
membrane. But the after an overnight run at 100 volts, the DNA was 
still in the plug.?????. So if any of you out there have any idea
please share them with me.
Many thanks
Ghis
P.S. I used low-melt agarose, but my pulse-field gel would be much
nicer if I could use a non-low-melt agarose.
--
Quand le sage montre la lune du doigt, l'idiot regarde le doigt

Ghislaine Allard
Departement de Biologie
Universite d'Ottawa



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