RNA extraction

cracker besco.1 at osu.edu
Tue Jul 18 15:36:49 EST 2000


I have found that if you use more Trizol in the initial extraction step,
your ratios will improve.  The lower ratios seem to work fine for things
such as RT-PCR, but, for me anyway, dont work as well in Northerns.....

Neil Saunders <s3003334 at pop3.unsw.edu.au> wrote in message
news:8ktl3v$atj$1 at mirv.comms.unsw.edu.au...
> > I have isolated total RNA from non adherent cells with the TRIZOL
reagent.
> > At the end of the procedure, I solubilized the pellet with DEPC-treated
> > water. After this, I measured OD 260 and 280 and calculated the ratio
> > 260/280. This ratio should be 1.6 to 2.0. Sometimes I have got around
1.6
> > but generally 1.4 to 1.5  ... it is too low but the RNA is OK on agarose
> gel
> > (no apparent degradation). Which step of the Trizol method can I improve
> to
> > increase this ratio? I have already read that to calulate the ratio it
is
> > better to take ODs of RNA in TE buffer but other people have already got
> > good ratios with RNA in water.
>
> If the RNA looks good on a gel, I wouldn't worry about the ratio.  What is
> the source of the RNA?  In my experience with Gram -ve bacteria
(Paracoccus
> denitrificans and E. coli), it's normal for the ratio to vary between 1.5
> and 1.8.  The theoretical ratio of 2 in all the manuals is just
> that-theoretical.  It depends on the base composition and other factors eg
> in some Gram -ves, the 23S cleaves into 2 fragments, but this isn't
> indicative of general degradation.
>
> Neil
>
> --
> School of Microbiology & Immunology,
> University of New South Wales,
> Sydney 2052,
> Australia
>
> Ph: +61 2 9385 2093
> Fx: +61 2 9385 1591
> email: neil.saunders at unsw.edu.au
> http://www.crosswinds.net/~nfws/index.htm
> ArchaeaWeb
> http://www.crosswinds.net/~nfws/archaea/index.htm
>
>
>







More information about the Methods mailing list