prepare DNA from flowering plant Azalea (rhododendron, Ericaceae)
Mike Prigge
prigge at darkwing.uoregon.edu
Wed Aug 30 19:52:58 EST 1995
I've had better luck getting clean plant DNA by precipitating using
the CTAB. (I guess when the salt conc gets too low, CTAB precipitates
carrying nucleic acids but not other saccharides with it.) To do this
follow Dilip's protocol up until BEFORE the isopropanol precipitation
then do the following:
Add 1/10 Volume [10% CTAB, 0.7 M NaCl] (heat to 50-65 degrees to make
less viscous)
Repreat CHCl3 extraction
Add 2 Volumes [1% CTAB, 50 mM NaCl, 10 mM EDTA], mix, let sit for 30
minutes.
Spin down pellet and resuspend in about 500 ul 1 M NaCl (heat tubes to
55 degrees if necessary) and transfer to microfuge tube
Add 1000 ul Ethanol to precipitate, invert several times, and pellet DNA
Wash pellets twice with 70% Ethanol, remove all the ethanol and
resuspend in 400 ul TE
Add RNAse and incubate at 37 - 50 degress for an hour
Extract with phenol:chloroform twice chloroform once
Add 40 ul 3M NaOAc and 1000 ul ethanol to precipitate
pellet then resuspend in TE (We use 100-400 ul for 5 grams of plant
tissue)
I found this in Methods in Arabidopsis Research edited by Koncz, Chua,
and Schell
Good Luck
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