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 1mL 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 750µL chloroform:isoamylic alcohol (24:1)

10. Centrifuge at 14000rpm for 5 min at 4oC.