RNA Extraction Protocol: Difference between revisions
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==Day 1== | ==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) | ::(''stop solution'' = 5% buffer equilibrated phenol [pH 7.4] in ethanol) | ||
3. Add sample up to 50 | |||
3. Add sample up to 50 ml | |||
4. Pellet sample by centrifugation at 5000 rpm for 10 min at 4°C | 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) | :::*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%) | :::*35 μl of SDS (20%) and 35 μl of sodium lauryl sarkosine (20%) | ||
:::*750 μl of phenol:chloroform:isoamylic alcohol (25:24:1) | :::*750 μl of phenol:chloroform:isoamylic alcohol (25:24:1) | ||
5. Remove samples from centrifuge. '''''Keep on ice!''''' | 5. Remove samples from centrifuge. '''''Keep on ice!''''' | ||
6. Discard the supernatant fraction and add 1 ml of extraction buffer to the pellet | 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) | ::(''extraction buffer = ''[10% CTAB in 2.5 M NaCl]:0.1 M phosphate buffer pH 5.8, 1:3) | ||
::Mix well and transfer | ::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) | 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 | 8. Centrifuge at 14000 rpm for 5 min at 4°C | ||
9. Transfer the upper aqueous phase into a new 2 mL tube | 9. Transfer the upper aqueous phase into a new 2 mL tube | ||
::Add 750µL chloroform:isoamylic alcohol (24:1) | ::Add 750µL chloroform:isoamylic alcohol (24:1) | ||
10. Centrifuge at 14000rpm for 5 min at 4oC. | 10. Centrifuge at 14000rpm for 5 min at 4oC. | ||
Revision as of 12:32, 22 May 2013
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 750µL chloroform:isoamylic alcohol (24:1)
10. Centrifuge at 14000rpm for 5 min at 4oC.
