RNA Extraction Protocol

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(Day 1)
(Day 2)
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==Day 2==
==Day 2==
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12. Cool RNA room centrifuge
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13. Centrifuge frozen tube at 14,000 RPM (18,000 g) for 1 hour at 4°C
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• Take Ambion DNase1 and buffer out of fridge and put on ice (starts thawing)
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14. Remove supernatant and add 500 μl of 75% ethanol
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15. Centrifuge at 18,000 g for 5 min at 4°C
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16. Remove supernatant and centrifuge at 18,000 g for 2 min at 4°C
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17. Remove any liquid residue from the tubes by pipetting
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Dry samples for 15 min at RT with caps off (evaporating alcohol)
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18. Pool every 2 samples (2-3 for LWS samples, 1 otherwise):
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Re-suspended in 100μl of  DNase I mix
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(Make one master tube containing: 90 μl ddH2O, 10 μl of 10xDNase buffer and 5 μl DNaseI per pool)
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19. Incubate at 37oC  for 30 min
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20. Add 350 μl Buffer RLT (from QIAGEN RNeasy kit)
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21. For LWS pooled samples, do the following twice:

Revision as of 17:58, 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 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 to allow full precipitation of nucleic acids.

Day 2

12. Cool RNA room centrifuge 13. Centrifuge frozen tube at 14,000 RPM (18,000 g) for 1 hour at 4°C • Take Ambion DNase1 and buffer out of fridge and put on ice (starts thawing) 14. Remove supernatant and add 500 μl of 75% ethanol 15. Centrifuge at 18,000 g for 5 min at 4°C 16. Remove supernatant and centrifuge at 18,000 g for 2 min at 4°C 17. Remove any liquid residue from the tubes by pipetting Dry samples for 15 min at RT with caps off (evaporating alcohol) 18. Pool every 2 samples (2-3 for LWS samples, 1 otherwise): Re-suspended in 100μl of DNase I mix (Make one master tube containing: 90 μl ddH2O, 10 μl of 10xDNase buffer and 5 μl DNaseI per pool) 19. Incubate at 37oC for 30 min 20. Add 350 μl Buffer RLT (from QIAGEN RNeasy kit) 21. For LWS pooled samples, do the following twice:

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