Solutions
(All solutions in RNAse free water! )

1. Extraction buffer (EB): for 50 ml

2% CTAB (hexadecyltrimethylammonium bromide) 1 g;
2% PVP (polyvinylpyrrolidone K30) 1 g;
100 mM Tris-HCl(pH 8.0) 5 ml [1M];
25 mM EDTA 2.5 ml [0.5M];
2.0 M NaCl 5.844 g;
0.5 g/L spermidine (25 mg)
(Mix and autoclave);
2% β –mercaptoethanol (add just before use) 20 µl/ml;

2. Chloroform : Isoamyl alcohol 24:1

3. 10 M Lithium chloride: for 50 ml 21.2 g

4. SSTE: for 50 ml

1.0 M NaCl 2.922 g;
0.5% SDS 2.5 ml [10%];
10 mM Tris-HCl (pH 8.0) 0.5 ml [1M];
1 mM EDTA (pH 8.0) 100 µl [0.5M];
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Protocol:

1. Heat up 0.5 ml extraction buffer to 65 °C (without β–ME) in a water bath (used 50 mg of N2-grind plant tissue; 5 ml EB/ 1-2 gr plant material)

2. Just before use add 10 µl β–ME to 0.5 ml preheated EB and add this to 50 mg N2-grind tissue; mix by vortexing

3. incubate for 2-3 minutes at 65 °C

4. Extract 2 times with an equal volume (0.5 ml) of chloroform:isoamyl alcohol, separate phases by centrifuging at RT for 10 min @ 13.000 rpm. Spin longer if phases are not well separated (upper phase contains RNA)

5. Transfer supernatant to new tube and add ¼ volume (125 µl) of ice cold 10 M LiCL

6. Overnight precipitation at 4 °C

7. Harvest by centrifugation 13.000 rpm for 20 min, 4 °C

8. dissolve pellet into 250 µl SSTE (preheated to 37 °C, otherwise a precipitate forms)

9. Extract once with an equal volume (0.25 ml) of chloroform:isoamyl alcohol, 10 min at 13.000 rpm.

10. Add two volumes (0.45 ml) of ice cold 95% ethanol to the supernatant, precipitate for at least 2 hours at -20 °C.

11. Spin 13.000 rpm for 20 min in a microcentrifuge to pellet the RNA. Remove supernatant and dry pellet.

12. Resuspend pellet in 50 µl RNAse free water

13. Measure on Nanodrop and check 250 ng on 1% agarose gel