The best CHEF electrophoresis conditions??

[Vivian Miao]

In article <1993Jun13.202917.12175 at gserv1.dl.ac.uk>, N.SALAZAR at lshtm.ac.uk
(Nelson Salazar) wrote:

(stuff deleted)
> how can I "predict" the best CHEF conditions
> to get a nice resolution of these "chromosomes".

> The E. histolytica "chromosomes" are about 0.3 to over 3 MegaBases in
> size. Another thing that is worrying me a lot, is the fact that I am
> getting a smear of DNA and therefore this makes very difficult to see
> the bands when stained with EtBr if there are any at all !!

Running your samples with standards will let you know approximately
 what sizes, how many species, etc, are in your prep.  If your estimate
 for E. histolytica is correct, and the chromosomes aren't  somehow
 physically unusual (e.g. circular), then conditions for the  Saccharomyces
 cerevisiae karyotype will be informative.   S. cerevisiae
 chromosomes cover most of the range you suggest for E. histolytica.
  For those larger than 2 megabase or so, you will need something like
 Schizosaccharomyces pombe, or Candida as size standards
 (Apergillus and Neurospora crassa chromosomes can be used if you
 need even larger size standards - hopefully not, though!)  Most, if not
all,
 of these are available commercially.    

  Although you can always try making your own plugs (fun with fungi!), I 
would recommend that you buy some yeast standard from a supplier, (who
 did you purchase the CHEF rig from?) just to get things moving along.
 If you choose to make your own standards, be sure
 you make them from a characterized strain - there are strain to strain
 differences in electrophoretic karyotypes.  One advantage to getting a
commercial prep is that if standards are resolved, but your
 sample is still smeary, then you should  backtrack and consider trouble
 shooting the chromosome prep protocol, rather than the running conditions.

I guess other folks will probably have more suggestions for you.  I haven't
 done this for a while, but I can vividly remember the sinking "oh no, not
 another smeary gel !" feeling.  (But don't worry, there is life after
CHEF!)

Good luck,
Vivian