Why can't I get agarose embedded HMW DNA to digest ?

[Todd DeZwaan]

In article <3kbn92$mfk at is.bbsrc.ac.uk>, cavell at BBSRC.AC.UK) (A Cavell) wrote:

> I am trying to do some long range physical mapping of the Brassica genome.
> 
> I obtain high moleclar weight DNA by embedding protoplasts in low melting
> 
> point agarose. These are then lysed and washed extensively using PMSF.
> 
> I follow standard protocols for digestion with rare cutting REN's but 
> 
> there seems to be a problem with diffusion of enzyme into blocks as I 
> 
> get little or no digestion.
> 
> Any Ideas ??

Have you tried incubating your agarose slabs containing your DNA on ice in
the presence of the enzyme and buffer for a couple of hours and then
shifting the slabs to a room temp water bath for gentle digestion of your
DNA?  This is the technique I have used in constructing yeast artificial
chromosome libraries and it has always worked well for me. I was not using
rare cutters though I was just trying to get partial digestion with a
six-cutter such as EcoRI. Good luck.