Materials and Supplies
Mortar and pestle
Tris EDTA buffer (ACROS Organics: DNase, RNase, and Protease free, ready to use,
pH 8.0, Cat # AC32734-5000)
5:1 Acid Phenol: Chloroform (pH 4.5, Cat #9720)
Saturated Phenol (pH 6.6, Cat# 9712)
High Salt (DEPC treat): 0.8M Sodium Citrate, 1.2M NaCl
- Clean surfaces with RNase Awayâ
- Take 8 kernels. Grind thoroughly until it is at the consistency of powder and store at -80oC
- Pipette 0.75mL of phenol into 15ml tubes
- Add 1 scoop of grinded tissue sample to the 0.75mL phenol. Homogenize 4min
- Add 0.75mL tris buffer to the 0.75mL phenol. Vortex 2min.
- Centrifuge at 14K RPM, 4oC, 10min.
- Without disturbing the interface, transfer the aqueous layer (top layer) to another 1.5ml tube containing 0.75ml of Phenol: Chloroform. Vortex 2min and spin at 14K RPM, 4oC, 10min.
- Without disturbing the interface, transfer the aqueous layer (top layer) for a 2nd time to another 1.5ml tube containing 0.75ml of Chloroform. Vortex 2min and spin at 14K RPM, 4oC, 10min.
- Carefully remove the aqueous layer to a 1.5ml tube. Add 250ul isopropanol (mix) and 250ul high salt (mix). Incubate at -20oC for 1 hour.
- Spin at 14K RPM, 4oC, 30 min.
- Decant supernatant, and wash with 75% ethanol.
- Spin at 6K RPM, 4oC, 6 min. Briefly dry pellet -6 minutes (not too long, or pellet will be extremely difficult to re-suspended).
- Re-suspend in 60ul DEPC water with 1ul RNasin
RNA can be further purified using the Qiagen RNeasy cleanup protocol