restriction cutting Chlamy DNA - summary

Wed Aug 31 19:16:56 EST 1994

Dear Chlamy types,
  A few weeks ago I asked for suggestions on why our lab might
be having huge problems cutting genomic DNA from some Chlamy
strains with enzymes that normally aren't too fussy, particularly
EcoR1 and Sal1.  Thanks to the many people who answered.  The
most common response was that our DNA preparations might be
contaminated with polysaccharides, apparently a common problem
with preps from plant tissues too.  Among the possible remedies
I tried CTAB precipitation, mostly because it's easy and cheap,
and we do lots of DNA preps.  The method I got was from Current
Protocols in Molecular Biology:  after a phenol-chloroform
extraction, add to the supernatant 1/7 volumes of 5M NaCl, mix VERY
well (otherwise the DNA precipitates), add 0.1 volumes CTAB solution
(10% in 0.7M NaCl), and extract with an equal volume of 24:1
chloroform:isoamyl alcohol.  This has totally eliminated our
         Cliff Zeyl
         b7jm at musicb.mcgill.ca

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