RNA precipitation with LiCl
Roderic at biogene.co.uk
Wed Jun 18 16:56:41 EST 1997
In article <Pine.A188.8.131.520612172451.41578A-100000 at asterix.uni-
muenster.de>, Andreas Vogel <vogela at uni-muenster.de> writes
>i´m isolating RNA from bean seeds using various protocols which utilize a
>precipitation in 3M LiCl as the final step. My pellet becomes very big and
>white and could not be cleaned by several washing steps with 70% EtOH. Does
>anybody know how to avoid this coprecipitation (of LiX?) or to clean the
Common contaminants of RNA purified in this fashion from plant tissue
are polysaccharides and traces of DNA.
Try the following
Disperse in 2M LiCl, 50mM EDTA and repellet
This will reduce DNA contamination
Disperse in 3M NaAc pH5.2 and repellet
This will reduce polysaccharide contamination, and small RNAs such as
tRNA. (I'm assuming you are interested in mRNA)
As a final cleanup the following can be performrd
Dissolve remaining pellet in volume "x" of 2%KAc by dispersal and
incubation at 55oC for 10'
Pellet insoluble material (junk)
Remove supernatant to a clean tube and add 2.5"x" EtOH
Mix and ncubate at -70oC for 15'
If you want the best quality RNA try one of the following which are more
costly, or require an ultracentrifuge
Anion-exchange chromatography after the 2M LiCl wash step
Ultracentrifugation with CsCl.
Roderic Fuerst E-mail: Roderic at biogene.co.uk
Bio/Gene Ltd Voice: +44 (0) 1480 861 831
6 The Business Centre Fax: +44 (0) 1480 861 899
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