RNA Extraction From Endosperm SDS-Trizol combo
(based on a protocol by Joao Leiva, Ricardo Dante and David Holding)
SDS Extraction (based on Prescott and Martin 1987)
1. Add 100mg ground endosperm to a tube with 200ul RNA extraction buffer.
To make 200ml Buffer:
- 10ml 1M Tris
- 30ml 1MLiCl
- 2ml 0.5M EDTA
- 118ml H20
- DEPC treat, and autoclave
- add 40ml 5% SDS
2. Add 200ul of 1:1 Phenol-Chloroform, shake it well and place them on ice for 5 min. mix occasionally.
3. Transfer Solution to a Phase Lock tube (Eppendorf) (pre-spin the Phase Lock tubes for 30 sec at 12000g before adding solution. Never vortex Phase Lock tubes.
4. Centrifuge the samples for 10 min @ 10000g, 4 C.
5. Add 200ul of Phenol-Chloroform, Shake well
6. Centrifuge samples for 10 min at 10000g, 4 C.
7. Add 200ul of Chloroform, shake well, and place on ice for 5 min. Mix occasionally.
8. Centrifuge samples for 10 min at 10000g, 4 C.
- Pour the aqueous phase into a new Phase Lock tube (the organic phase will be locked in the bottom of the tube)
- Add 1 ml Trizol.
- Shake well for 15 sec. Incubate for 5 min at room temp.
- Add 200ul of Chloroform, shake it well and incubate for 2-3 min at room temp.
- Centrifuge for 10 min at 10000g, 4 C.
- Pipet 700ul of aqueous phase to a fresh 1.5ml microfuge tube.
- Add 700ul of Isopropanol, mix well, and incubate for 10 min on ice.
- Centrifuge for 10 min at 10000g, 4 C.(Precipitate can be seen at this point)
RNA Washing and Dissolving
- Pour out the supernatant
- Add 1 ml of 70% EtOH (in DEPC treated water)
- Centrifuge for 5 min at 10000g, 4 C.
- Carefully pour out the supernatant.
- Air dry the RNA pellet.
- Re suspend in 50ul of DEPC treated water