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Total RNA Isolation

Zürich Online Bibliothek Uni ZH Vorlesungen Institute of Plant Biology Home Page U of Zürich Home Page   Schneitz Lab Home Page / Methods / RNA Work / Total RNA Isolation
Total RNA Isolation
Reference:  Schneitz Lab Last updated: 2/1/00 By: Kay Schneitz

1.0 g plant tissue:

  1. grind tissue with pestle and mortar in liquid nitrogen
  2. transfer to a 50 ml falcon tube containing 10 ml of sol D
  3. add 1 ml 2 M NaOAc pH 4.1
  4. add 10 ml phenol
  5. add 2 ml chloroform/isoamylalcohole (49:1)
  6. mix thoroughly
  7. shake finally for 10 sec
  8. cool on ice for 15 min
  9. spin at 3500 rpm/4°C for 20 min in an Omnifuge 2.0 RS centrifuge (Heraeus)
  10. transfer aqueous phase to a fresh 30 ml corex glass tube
  11. add 10 ml isopropanole
  12. precipitate for 1 hour at -20°C
  13. spin at 8000 rpm/4°C for 20 min in a HB-4 rotor
  14. dissolve RNA pellet in 3 ml sol D (ev. heat to 60°C to dissolve pellet)
  15. add 3 ml isopropanole and precipitate for 1 hour at -20°C
  16. spin at 8000 rpm/4°C in a HB-4 rotor for 15 min
  17. resuspend pellet in freshly prepared 75% EtOH
  18. repeat centrifugation, remove supernatant and air dry for 10-15 min
  19. dissolve pellet in 3-5 ml of sterile, freshly autoclaved H2O (ev. heat to 60°C to dissolve pellet)


Guanidium thiocyanate sol:

100.0 g GTC

117.2 ml H2O

7.04 ml 0.75 M NaZit pH 7

10.56 ml 10% sarcosyl

220 ml total

Sol D:
add 0.36 ml b-mercaptoethanole/50 ml GTC sol (1.6ml/220 ml), stable for 1 month at RT


2 M NaOAc pH 4.1 (100 ml):

dissolve 27.2 g NaOAc in about 54 ml H2O and add about 46 ml conc HAc


As usual when working with RNA: pay attention, always wear gloves, use only freshly prepared and autclaved material and use sterile plastic pipettes. In our experience there is no need for DEPC-treated material.

Corex tubes and pestle and mortar have to be pretreated with H2O2 for 30 sec, rinsed several times with H2O and baken for 6 hours at 180°C in oven.


Reagent Supplier Cat.-#
guanidium thiocyanate Sigma G-9277
isopropanole Fluka 59300
NaOAc Fluka 71180
NaZit Sigma S-4641
sarcosyl Sigma L-9150
b-mercaptoethanole Sigma M-6250

  © Copyright 1997-2000, Kay Schneitz. Last updated 2/1/00; 12:25:11 PM. Send comments to the webmeister.