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RNA-isolation (TRIZOL method)

RNA-isolation (TRIZOL method)

 

Isolation of RNA from whole worms using TRIZOL reagent (Gibco-BRL).

1) Wash worms from a 60 mm plate with 1 ml DEPC-treated water.

2) Spin down worms at 4000xg in microfuge for 1 minute.

3) Remove excess water and add 1 ml Trizol reagent. Vortex and invert tube. Let sit at room termerature for 10 minutes (should see stringy-like material).

4) Spin tubes in microfuge at 14K for 10 minutes at 4° C.

5) Remove supernatant into fresh RNAse free eppendorf tube and add 200 µl of chloroform.

6) Vortex for 15 seconds and let sit at room temperature for 3 minutes.

7) Spin in microfuge at 12K for 15 minutes at 4° C.

8) Carefully remove the top layer (clear) into a new RNAse free eppendorf tube and add 500 µl of Isopropanol. Invert to mix. Let sit for 10 minutes at room temperature.

9) Spin in microfuge at 12K for 10 minutes at 4° C. (RNA will be seen as a nice white pellet).

10) Wash pellet with 100 µl of 75% ethanol (made by diluting into DEPC-treated water).

11) Spin in microfuge at 7500 xg for 5 minutes at 4° C.

12) Remove supernatant and air dry pellet for 10 minutes.

13) Dissolve pellet in 25 µl of DEPC-treated water. Heat for 10 minutes at 60° C to help dissolve RNA.

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compiled by Johanna O'Dell-Whitacre

Aroian Lab Protocols