Arabidopsis total RNA prep - medium scale

Adapted from Lagrimini et al (1987) PNAS 84: 7542

Per sample need: morter, pestle, spatula, 2-30 ml corex tubes (all baked), plus liquids:
XB, phenol, liquid nitrogen, chloroform, 3M NaAc (pH 5.3), ETOH, diH2O.

1. For each sample, label a 30 ml autoclaved Corex tube and add:
5.0 ml extraction buffer (XB)
5.0 ml H2O-saturated phenol
Per sample, put 1 mortar, 1 pestle and 1 spatula (autoclaved) on dry ice.
Get dewar with liq N2.

2. Grind 1-2 g frozen tissue finely in mortar.
(Add tissue to mortar, add a little liq N2, let evaporate and grind).
Important: max. 2 g., otherwise yield decreases.
Grind for a minute after tissue appears fine.

3. Transfer powder with cooled spatula to Corex tube.
Mix powdered plant material with XB/Phenol using spatula.

4. Add 5 ml CHCl3. Mix with spatula.

4a. Optional: Polytron sample for 45 sec at setting 5.5

5. Spin in swinging bucket rotor: 10 min at 6000 rpm
Sorvall HB-4, HS-4 or equivalent.

6. Transfer aqueous phase to new baked Corex tube,
record vol. on label and transfer label to new tube.

7. Add: 1/10th vol. 3M NaAc, pH 5.3 (autoclaved)
2 volumes 95% ETOH, -20° C
Parafilm and mix by inversion.
Incubate on ice for 30 min.

8. Spin as above: 10 min @ 6000 rpm.

9. Decant supernatent and drain pellet for 30 min.
Put tubes in rack at an angle pointing down; do not overdry.

10. Touch top of inverted tube to Kimwipe to grab residual drop of alcohol.
Add 4 ml ddH2O (autoclaved) to pellet. Vortex repeatedly and keep on ice. If neccesary, use 1 ml pipetman to resuspend. OK if small particles are left behind. Keep solution near tube bottom when manipulating.

11. Add 2.5 ml 8M LiCl (autoclaved). Parafilm tube, mix and refrigerate overnight.
Alternatively, place in -80 C freezer for 1 hour, then thaw on ice*.

12. Spin as above: 10' @ 6K rpm.

13. Rinse pellet with 1 ml 80% ETOH from -20° C freezer .
Spin briefly if pellet detaches from tube. Let pellet dry as above.

14. Resuspend pellet in 150 ul diH2O (autoclaved).
Keep on ice. Hold volume down (or you may have to dry vol. down to get conc. high enough later on). If any particulate material is present, spin tube and transfer super. to new tube.

17. Spec to quantitate.

18. Gel electrophoresis as described in RNA Prep Mini (link).

Materials:
Extraction buffer (XB; Prepare fresh):
for 100 ml:
4g 4% p-aminosalicylic acid (Sigma A-3505)
1g 1% 1,5-naphthalenedisulfonic acid, disodium salt (Kodak 2765)
Water-saturated phenol: Add ddH2O to solid phenol. Place in 65°C waterbath. When fluid, mix well, repeatedly. Store @ room temperature. Discard if it turns yellow.

*for details on use of LiCl to precipitate RNA, see: http://www.ambion.com/techlib/tb/tb_160.html

 

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