RNA PREP Trizol

(methode Invitrogen modifiée)

Wear gloves at all times

Prepare DEPC water by adding 0.1% DEPC in fume hood. Leave o/n and autoclave following day.

LiCl buffer; 4M LiCl, 20mM Tris pH 7.5, 10 mM EDTA made up with DEPC water and autoclaved.

Trizol reagent Gibco-BRL # 15596-026

1. Precool a mortar (which was in -20°C one night) with liquid Nitrogen. Add 1ml Trizol and grind to a powder

2. Filter 10ml o/n filamentous culture through miracloth or muslin and immediately put into liquid Nitrogen + trizol and grind to a fine powder adding more liquid Nitrogen as necessary.

3. Put ground mycelium into an eppendorf tube. Vortex 1min.

Keep at room temp 5min to allow dissociation of nucleoprotein complexes.

4. Spin 12000g 10 mins at 4°C. (Most ADN are eliminated). Transfer cleared supernatant to fresh tube.

5. Add 2/10 volume (200µl) chloroform and shake vigorously by hand 15 seconds.

Keep at room temp for 3 minutes.

6. Spin 12000g 5minutes at 4°C. (Upper colourless phase contains RNA).

7. Transfer colourless phase to fresh tube and add 0.25ml isopropanol and 0.25ml SSC (0.8M sodium citrate, 1.2M NaCl) (for 1ml de Trizol at the biginning). Keep samples at room temp 5 minutes.

8. Spin 10 mins 4°C 12000g (RNA pellet forms a gel-like ppt.)

9. Remove isopropanol carefully and wash pellet twice with 1ml 70% Ethanol (-20°C) spinning as above.

10. Remove ALL alcool by centrifugation and dissolve in 500µl DEPC water.

11. Add 1vol LiCl buffer. Ppt. at -20° at least 60minutes. (o/n is OK )

12. Centrifuge at maximum speed 30 minutes at 4°C.

Wash pellet twice with 70% Ethanol at -20°.

Remove ALL alcool by centrifugation and dissolve in DEPC water (50-100µl).

13. Run 1µl out on 1.2% TBE agarose gel to check quality of RNA