plant rna extraction : purity problems
Dr. Peter Gegenheimer
PGegen at UKans.nolospamare.edu
Tue Aug 1 13:13:25 EST 2000
On Mon, 24 Jul 2000 19:55:00, Sam Lievens <salie at gengenp.rug.ac.be> wrote:
ð hi all,
ð i'm having serious problems getting pure rna out of the roots of our
ð plant. it's a tropical legume. getting rna from the aerial parts is no
ð problem (od 260/280 of 2.0 and 260/230 of about 2.3), pure enough to
ð prepare cdna of.
ð so two questions: how can i get pure rna and how can i further purify
ð the dirty rna i already have (i already precipitated it again with licl,
ð doesn't help; i did 2 fenol/chloroform extractions and ethanol
ð precipitation, doesn't help neither).
1- The polysaccharide shouldn't interfere with anything *IF* it is uncharged
(non-ionic). (For example, RNA or DNA co-precipitated with glycogen will have
this absorbance profile, but is "clean.") Unfortunately, many plant
polysaccharides seem to be negatively-charged, and will inhibit enzymes that
bind nucleic acids. Have you tried working with your "dirty" RNA?
2- Another poster suggested the use of CTAB (cetyltrimethylammonium bromide).
This will separate nucleic acids from un-charged polysaccharides, but I'm not
sure about charged polysaccharides. If you try it, remember that there are two
"CTAB" protocols. One uses CTAB simply as an ionic detergent to lyse cells and
denature protein. The lysate is then extracted with phenol or chloroform. The
original protocol, and the one you want, uses CTAB to selectively precipitate
the nucleic acids. As I recall, the CTA(+) -- RNA(-) complex is soluble at
high [NaCl~0.7M, and precipitates when you dilute it to low [NaCl]. If you
can't find this in a plant protocol book, search on Murray & Thompson who
published the protocol in the 1980's. (David Sabatini originally developed in
in ~1970 for precipitating nanogram amounts of DNA.)
3- There are protocols (not at my fingertips) in which the crude plant nucleic
acids are pelleted through a CsCl cushion in an ultracentrifuge, or perhaps
even in a microfuge! This will pellet RNA and leave polysaccharides in
solution. It's certainly worth a try to clean up the samples you already have.
| Dr. Peter Gegenheimer | Vox: 785-864-3939 FAX: 785-864-5321 |
| Department of | PGegen at UKans.nospam.edu |
| Molecular Biosciences | http://rnaworld.bio.ukans.edu/ |
| University of Kansas |"When you have excluded the impossible, |
| 2045 Haworth Hall | whatever remains, however improbable, |
| Lawrence KS 66045-2106 | must be the truth." S. Holmes |
More information about the Methods