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MOLECULAR BIOLOGY: WORKING WITH DNA

ISOLATION - RNA

Arabidopsis RNA Extraction

Arabidopsis RNA Extraction Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

This method routinely yields 200 μg RNA per gram of tissue.

Procedure Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

1. Freeze 10 g of tissue in liquid nitrogen and grind to a fine powder with a mortar and pestle. Put the powder into a 50 ml disposable centrifuge tube. 2. Thaw the tissue in 25 ml of Extraction Buffer and 5 ml of 10% SDS. Homogenize in a Polytron homogenizer until no large pieces of tissue remain. 3. Centrifuge at 2,500 X g (4,000 rpm in a Sorvall RC3C rotor) for 10 min to pellet the debris. 4. Transfer the supernatant to a fresh 50 ml polypropylene tube. Top the tube off with phenol and shake briefly. 5. Centrifuge at 2,500 X g (4,000 rpm in a Sorvall RC3C rotor) for 5 min. 6. Transfer upper phase to a clean centrifuge tube. 7. Repeat Steps #3 to #5 twice using a Phenol/Chloroform extraction and then once with a Chloroform extraction. 8. Add 16 ml of 5 M Lithium Chloride keeping the final Lithium Chloride concentration greater than 2 M. 9. Mix and precipitate the RNA overnight at 4°C in siliconized tubes. 10. Centrifuge for 10 min at 4,300 X g (6,000 rpm using a Sorvall SS34 rotor) at 4°C. 11. For total RNA, resuspend in an appropriate volume of 0.2 M Lithium Chloride and precipitate in 2.5 volumes of 100% Ethanol. 12. Centrifuge for 10 min at 4,300 X g (6,000 rpm using a Sorvall SS34 rotor) at 4°C. 13. Discard the supernatant. 14. Resuspend the total RNA pellet in TE or store in 70% Ethanol at -70°C until ready for analysis.

Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

TE    10 mM Tris pH 8.0 1 mM EDTA 70% (v/v) Ethanol 0.2 M Lithium Chloride 5 M Lithium Chloride 1:1 Phenol:Chloroform    CAUTION! See Hint #1 10% (w/v) SDS Extraction Buffer    50 mM Tris, pH 8.0 5 mM EDTA 150 mM NaCl

BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

Tris Chloroform Phenol Nitrogen, Liquid Sodium Chloride Ethanol SDS Lithium Chloride EDTA

Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.