Megabase DNAs and PFGE?

[Dan Riggs]

<x-flowed>Hello All:

We are interested in using pulsed field gel electrophoresis to separate
large fragments (approx. 0.2 to 1Mbp) of Arabidopsis DNA.  A look
through the databases reveals that very little has been published on
this. Can anyone provide practical advice on the following questions:

1. How does one prepare megabase-sized DNA?  Do standard leaf
protoplast/agarose embedding procedures work well?

2. Does anyone have any experience in using restriction enzymes which
cut infrequently (and preferably not at the whim of methylation?); has
anyone tried homing endonucleases?

3. Any advice for PFGE parameters?

Thanks for your help!

Dan Riggs

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