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        Total RNA Isolation

        互联网

        1011

         

        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 H2 O (ev. heat to 60°C to dissolve pellet)

        Solutions:

        Guanidium thiocyanate sol:

        100.0 g GTC

        117.2 ml H2 O

        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 H2 O and add about 46 ml conc HAc

        Remarks:

        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 H2 O2 for 30 sec, rinsed several times with H2 O and baken for 6 hours at 180°C in oven.

        Materials:

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

         

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