RNA Extraction Protocol
Extraction Protocol modified from Griffiths et. al, 2000
Can also be used for DNA extraction by increasing phosphate buffer pH to ~8
Change gloves often and use fume hood when working with phenol!
Day 1
1. Start centrifuge cooling with 50 mL tube holders
2. Add 5 ml of cold stop solution to a fresh 50 mL tube
- (stop solution = 5% buffer equilibrated phenol [pH 7.4] in ethanol)
3. Add sample up to 50 ml
4. Pellet sample by centrifugation at 5000 rpm for 10 min at 4°C
- While centrifuging, prepare 2 ml screw-cap tubes (2 tubes per sample) with:
- 0.5 g of 0.1 mm zirconia-silica beads (Biospec products)
- 35 μl of SDS (20%) and 35 μl of sodium lauryl sarkosine (20%)
- 750 μl of phenol:chloroform:isoamylic alcohol (25:24:1)
5. Remove samples from centrifuge. Keep on ice!
6. Discard the supernatant fraction and add 1 ml of extraction buffer to the pellet
- (extraction buffer = [10% CTAB in 2.5 M NaCl]:0.1 M phosphate buffer pH 5.8, 1:3)
- Mix well and transfer 1 ml aliquots into prepared 2 ml screw-cap tubes
7. Homogenise in a mini-beater (Biospec products) for 2 min (3 min in less powerful beater)
8. Centrifuge at 14000 rpm for 5 min at 4°C
9. Transfer the upper aqueous phase into a new 2 ml tube
- Add 750 µl chloroform:isoamylic alcohol (24:1)
10. Centrifuge at 14000 rpm for 5 min at 4°C.
- While centrifuging, prepare tubes for precipitation (these will go in -80°C freezer)
- 5 ul 0.5M MgCl2
- 75 ul 3M sodium acetate
11. Transfer supernatant to prepared tubes and add 800µL ice-cold isopropanol
12. Incubate overnight at -80°C.