Help: genomic DNA purification
ckwen at expert.cc.purdue.edu
Fri Aug 9 12:40:40 EST 1996
Hongyu yang (yang at tifton.cpes.peachnet.edu) wrote:
: Hi netters:
: We were able to isolate genomic DNA using 2xCTAB protocol from plant leaves. Following
: isopropanol addition there was a big cloud of stuff (we thought it was the DNA
: precipitation). We dried the "DNA pellet" and tried to redisolve it in TE, some gelly
: stuff could not be disolved even with 65C water bath.
: I'll appreciate any advices to remove the "gelly stuff" from my DNA solution.
As my experiences with "difficult plants", never use IPA to precipitate the
nucleic acids after the CTAB/chloroform extraction! A lot of polysaccharides
or proteins may precipitate along with the nucleic acids. You may use a 1xCTAB
with NaCl conc of 1M to do thee extraction, then two or three chloroform ext.
Add CTAB precipitation buffer, which is CTAB in TE, to adjust the NaCl conc.
to below 0.35 to 0.3M. Stand at RT for greater than 30min, spin down the
nucleic acids. Most of your polysaccharides/proteins would be removed. Some
positively/neutrally charged molecules could stay. You may not be able to
get 100% pure nucleic acids by most of the current methods, anyway, and CTAB
is the best methods I have used so far.
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