need help w/ phage DNA digestion!

Jim Owens jow at helix.nih.gov
Fri Nov 18 08:34:25 EST 1994


In article <3ah64n$koj at noc.usfca.edu> Mandalaparthy,
padmmv00 at ac.usfca.edu writes:
>great help. The Dna prep is pretty clean and I did a couple of
>Phenol/chloroform  extractions and reppted the DNA w/ sodium acetate.I
>tried incubating overnight aswell .

Two suggestions:

Make sure the EcoRI is working by doing a parallel digestion on lambda
phage DNA.

Make sure there are no traces of phenol and chloroform.  You might try
dialysis for an hour or two before digesting with the restriction enzyme.
 If the volume is small, up to 200ul, dialyse by applying to the shiny
side of a VMWP 02500 Millipore filter floating on a petri dish of TE.

Good luck,

Jim Owens



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