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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