Friday, November 07, 2003

Description
Methods for Plant RNA Isolation

Procedure
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).

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