| |
| 1. |
|
Combine 5 ml extraction buffer and 5 ml phenol (DEPC H2O equilibrated) to a sterile 50 ml tube. Heat to 80° for at least 10 minutes. |
| 2. |
|
Add well ground tissue (1-2 grams) to hot phenol/extraction buffer. |
| 3. |
|
Vortex 30 sec. Add 5 ml chloroform/isoamyl alcohol (24:1) and vortex 30 sec. . |
| 4. |
|
Centrifuge in a superspeed centrifuge for 25 min, 9000 rpm, at 4°. |
| 5. |
|
Remove aqueous phase to a clean sterile 50 ml tube. Add an equal volume of 4M LiCl. |
| 6. |
|
Freeze at -80° for at least 1 hour. |
| 7. |
|
Centrifuge 20 min, 9000 rpm, 4°. |
| 8. |
|
Pour off supernatant and wash pellet with 2-5mls 70% EtOH. Spin at 4° for 10 min. |
| 9. |
|
Pour off EtOH and air dry pellet. |
| 10. |
|
Resuspend pellet in 50 - 200ul ddH2O |
| |
|
|
| |
|
RNA Extraction Buffer
100mM LiCl
100 mM Tris pH 8.0
10mM EDTA
1% SDS
From the laboratory of James J. Giovannoni USDA Plant, Soil, and Nutrition Laboratory Boyce Thompson Institute for Plant Research Tower Road, Ithaca, NY |
