Holmes, Dawn 1-2
RNA Extraction from Cells using TPE and Linear Acrylamide
1. Resuspend pellet from a 50 mL conical in 3 mL cold (4˚C) TPE buffer (100 mM
Tris-HCl, 10 mM EDTA, 100 mM KH2PO4, pH 8.0)
2. Transfer 375 μL to each of 8 tubes.
3. Add 100 μL Plant RNA isolation aid (Ambion #9690)
4. Add 1 mL cold acetone (stored at -20˚C.)
5. Mix manually ~20 times.
6. Centrifuge on high for 5 minutes.
7. Discard supernatant
8. Add 2 μL RNase inhibitor (Ambion Superase)
9. Resuspend in 1 mL DEPC water (Ambion)
10. Add 10 μL lysozyme (50mg/mL)
11. Add 3 μL Proteinase K (20mg/mL)
12. Add 30 μL 10% SDS.
13. Incubate at 37˚C for 10 minutes.
14. Centrifuge on high for 15 minutes.
15. Remove supernatant to a new tube
16. Add 50 μL Plant RNA isolation aid
17. Add 600 μL hot (70˚C) acidic phenol (pH 4.5, Ambion)
18. Add 400 μL chloroform:isoamyl alcohol.
19. Mix on rotator for 10 minutes.
20. Centrifuge on high for 5 minutes.
21. Remove aqueous phase to a new tube
22. Add 600 μL hot acidic phenol
23. Add 400 μL chloroform:isoamyl alcohol.
24. Mix on rotator for 5 minutes.
25. Centrifuge on high for 5 minutes.
26. Add 100 μL 5N ammonium acetate to an empty tube.
27. Add aqueous phrase
28. Mix well
29. Add 4μL linear acrylamide (5mg/mL)
30. Mix.
31. Add 1 mL isopropanol (stored at -20˚C)
32. Vortex.
33. Precipitate RNA at -20˚C for 1 hour.
34. Centrifuge on high for 30 minutes.
35. Discard liquid.
36. Add 750 μL cold 70% ethanol (stored at -20˚C)
37. Vortex.
38. Centrifuge on high for 30 minutes.
39. Dump ethanol and get rid of rest with P200.
40. Air dry pellet at room temperature for about 8 minutes
41. Resuspend in 30 or 50 μL DEPC water
42. Vortex
43. Let sit 15 minutes at room temperature.
Holmes, Dawn 2-2
Cleanup with RNeasy RNA Cleanup kit (Qiagen):
1. Combine 2 or 4 tunes into one (~100 μL.)
2. Add 350 μL RLT buffer
3. Make fresh RLT buffer with 10 μL β-mercaptoethanol per 1 mL
4. Mix by pipetting.
5. Add 250 μL room temperature 100% ethanol.
6. Mix by pipetting.
7. Transfer to spin column
8. Centrifuge at 10,000 rpm for 15 seconds.
9. Transfer to column to a new 2 mL tube.
10. Add 500 μL RPE buffer.
11. Centrifuge at 10,000 rpm for 15 seconds.
12. Dump eluant and add another 500 μL RPE buffer.
13. Centrifuge on high for 2 minutes.
14. Transfer column to a new 1.5 mL tube.
15. Centrifuge on high for 1 minute.
16. Transfer column to a new 1.5 mL tube.
17. Add 50 μL DEPC water
18. Centrifuge at 10,000 rpm for 1 minute.
19. Suck up eluant and rerun over column again.
20. Centrifuge at 10,000 rpm for 1 min.
DNase Treatment:
1. Combine 2 tubes for 100 μL RNA
2. Add 2 μL DNase
3. Add 10 μL buffer
4. Incubate 37˚C for 30 min.
5. Add 20 μL resin, let sit at room temperature 5 minutes.
6. Centrifuge on high for 1 minute.
7. Remove RNA supernatant to a new tube.
TPE Buffer, pH 7.6 (50mL):
5ml of 1M Tris
1ml of 0.5M EDTA
5ml of 1M KPO4 pH 7.6
39ml of Milli-Q water
7M KPO4 pH 7.6 (500mL):
430ml of 1M K2HPO4
70ml of 1M KH2PO4