Good mini-prep for genomic DNA

chlamy at ACPUB.DUKE.EDU chlamy at ACPUB.DUKE.EDU
Fri Dec 3 11:32:57 EST 1993


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





More information about the Chlamy mailing list