suggested rapid genomic micropreps

daemon at net.bio.net daemon at net.bio.net
Tue Jan 13 22:43:39 EST 1998


Yesterday I posted a request for a quick and tiny genomic microprep
suitable for PCR on a few embryos.  THANKS to all those who responded.
Some folks requested that I post the responses.  People sent a diversity
of methods, ranging from grinding in Tris extraction buffers with teflon
pestles, to simply sitting on embryos with your keys in your back
pocket.  Below are 4 representative methods, with their authors.
  - Dan Vernon

   Department of Biology
   Whitman College
   Walla Walla, WA 99362

1)  Plant material was macerated with a Teflon pestle (VWR) for 5
seconds in a
1.5-ml microcentrifuge tube, then further macerated after the addition
of
0.5 ml of extraction buffer (200 mM Tris pH 7.5, 250 mM NaCl, 25 mM EDTA
pH
8, 0.5% SDS).  Solids were removed by centrifugation at 16,000 x g for
10
min.  Nucleic acids were precipitated with an equal volume of
isopropanol
then resuspended in 50 microliters of 10 mM Tris pH 7.5, 1 mM EDTA pH
8.
Use 2 microliters for a 30 ul PCR reaction.  Spin the
samples for a few min. before taking the 2 ul as this pellets some  crud
which can interfere with the PCR.
This method was used to isolate genomic DNA from 1 to 30 imbibed seeds,
fresh and dried seedlings, cotyledons, young and old leaves, flowers and
siliques.  The preparations are stable for at least a year at -20 =B0C.
  [this from an article recently submitted to The Plant Journal]
Sincerely,
  Michael Neff, PhD
  The Salk Institute for Biological Studies


2)  We have had good success just grinding a small amount of leaf tissue
in TE, spinning out the debris in a  microfuge and using the
supernatant (5 ul in a 50 ul reaction).
  Tom Mcknight

3)  Boil the half dead embryos in a small amount of water (50 -100 ul)
for 10
mintues.  Use the water for setting up PCR reactions.  The amount needed
depends on your primers.  Grinding the boiled tissue with a pellet
pestle
sometimes helps.
  David A. Patton
  Novartis Crop Protection, Inc.

4) The prep described by Edwards et al. in NAR works quite well in our
hands
for PCR on limited amount of tissue. We routinely do the standard prep
(protocol and reference on our web site (http://billie.harvard.edu) on
single kanamycin sensitive seedlings that have been selected against on
MS + kan for both CAPS and SSLP reactions. For very small amounts of
tissue it is worth adding a little carrier tRNA to the isopropanol
precipitation to make sure you bring down the DNA. We have done a
modified prep (basically using less extraction buffer and resuspending
in
a smaller volume) on single fertilized ovules 48 hours post-pollination
and have no trouble detecting the paternal contribution to the genome.
    Robert E. Pruitt  (pruitt at billie.harvard.edu)






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