Chlamydomonas Mini Whole Cell DNA Prep.
Originally developed by Scott Newman in Boynton-Gillham lab. I'm told they
now grow 1 ml aliquots of cells in multiwell plates rather than working
from patches on agar.
-Scrap cells off plate into 1.5 ml Eppendorf tube that contains 0.5 ml TEN
buffer.
-Resuspend vigorously by vortexing, spin for 10 sec. and aspirate off
supernatant.
-Resuspend cells in 150 ul H2O on ice and add 300 of SDS-EB buffer, vortex
to mix.
-Extract once with 350 ul phenol/CIA(1:1) for few min by vortexing,
separate phases by centrifugation for 5 min., transfer aq. phase to a new
tube.
-Extract once with 300 ul CIA (24:1), transfer aq. to a new tube.
-Add 2 volumes abs. ethanol, incubate on ice for 30 min., centrifuge for 10
min., wash pellet once with 200 ul 70% ethanol.
-Dry pellet and resuspend in ca. 40 ul H2O, for Southern analysis use about
1-3 ul.
TEN=10 mM Tris-Hcl, 10 mM EDTA, 150 mM NaCl.
SDS-EB= 2% SDS, 400 mM NaCl, 40 mM EDTA, 100 mM Tris-Hcl, pH 8.0.
Elizabeth Harris
chlamy at acpub.duke.edu
