Methods for Plant RNA Isolation

[KRISTJANNSON at NCCCOT7.AGR.CA]

Hi netters,

We use an AMES/Chloroform extraction in our lab to extract total RNA from
potato leaves for viroid analysis. The protocol is as follows:

AMES Buffer (for 200mL):	11.7g NaCl
				160 mL dH2O
				Adjust pH to 6.0
				0.40g MgCl2
				8.21g Na-acetate
				40mL EtOH
				6.0g SDS
We homogenize the plant material(usually leaves) and dilute the sap 1:5 in AMES
We then incubate for 30min. @37oC followed by the addition of an equal vol.of
chloroform.Vortex and store on ice. We then centrifuge the samples @ 3000rpm
in a Sorvall ss-34 for 10min. Total RNA is contained in the upper aqueous layer.
This protocol works well for us. We use it to extract total RNA for analysis
with DIG-labelled cRNA probe for PSTVd (Potato Spindle Tuber Viroid).

Hope this helps.
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Andrew Scott		Agriculture Canada - Central Plant Health Laboratory

3851 Fallowfield Rd.				Phone:(613)998-9320 X5904
Nepean, Ontario CANADA				Fax:(613)996-2497
K2H 8P9					Email:Kristjannson at ncccot7.agr.ca
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