RNA Extraction Protocol: Difference between revisions
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''Can also be used for DNA extraction by increasing phosphate buffer pH to ~8'' | ''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!'' | ''Change gloves often and use fume hood when working with phenol!'' | ||
==Day 1== | ==Day 1== | ||
# Start centrifuge cooling with 50 mL tube holders | |||
# 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. | |||
Revision as of 16:52, 20 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
- Start centrifuge cooling with 50 mL tube holders
- 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)
- While centrifuging, prepare 2 ml screw-cap tubes (2 tubes per sample) with:
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.
