Holmes, Dawn 1-2
RNA Extraction from Sediment/Electrode/Filter using TPE, tRNA, and Linear
Acrylamide
Always Keep on Ice
1. In 2 mL screw cap tubes:
a. Weigh out 0.5 g sediment
b. Fill ½ way with scrapped Electrode
c. Fill tube ¾ full with crushed filter.
2. Add 500 mL Tris-phosphate EDTA buffer (100 mM Tris HCl, 10 mM EDTA,
100 mM KH2PO4 pH 8.0) or increase to 800 mL for Electrode.
3. Sometimes use 1 ml RNA protect + 0.5 g sediment
4. Spin for 5 minutes to make pellet if needed
5. Add 100 µl Plant RNA Isolation Aid.
6. Add 1 ml cold acetone (stored at -20˚C).
7. Mix manually ~20 times.
a. Bang upside down to unstick pellet if needed
8. Centrifuge on high for 5 minutes.
9. Discard supernatant and add 2 µl RNase inhibitor (Ambion Superase)
10. Resuspend in 1 ml DEPC water (0.1%)
11. Add 10 µl lysozyme (50 mg/ml)
12. Add 3 µl Proteinase K (20 mg/ml)
13. Add 30 µl 10% SDS.
14. Incubate at 37˚C for 10 minutes. (Exact timing)
15. Centrifuge on high for 15 minutes.
16. Remove and save supernatant
17. Add 50 µl Plant RNA Isolation Aid (Ambion)
18. Add 10 µl tRNA (yeast from Sigma as carrier)
19. Add 600 µl hot (70˚C) acidic phenol (pH 4.5 from Ambion
a. Doesn’t pipet accurately fill to line
20. Add 400 µl chloroform:isoamyl alcohol.
21. Mix on rotator for 10 minutes.
22. Centrifuge on high for 4 minutes.
23. Remove and save aqueous phase
24. Add 600 µl heated acidic phenol (pH 4.5)
25. Add 400 µl chloroform:isoamyl alcohol.
26. Mix on rotator for 5 minutes.
27. Centrifuge on high for 5 minutes.
28. Add 100 µl 5 N ammonium acetate to empty tube
29. Add aqueous phase from step 27 to same tube.
30. Mix well
31. Add 4 µl linear acrylamide or 20 μl glycogen
32. Add 1 ml isopropanol
33. Vortex
34. Precipitate RNA at -20˚C for ½ hour - 1 hour
35. Centrifuge on high for 30 minutes.
36. Discard liquid
Holmes, Dawn 2-2
37. Add 750 µl 70% ethanol
38. Vortex
39. Centrifuge on high for 10 minutes
40. Remove all ethanol with a P200
41. Air dry pellet for 5 min.
42. Resuspend pellet in 50 mL DEPC water
43. Vortex
44. Let sit 15 minutes.
Clean up RNA with Rneasy RNA Cleanup kit (Qiagen):
1. Combine 2 of the tubes into one tube (100 μl)
2. Add 350 µl RLT buffer (with β-mercaptoethanol added- make upRLT buffer, add
10 µl mercaptoethanol to 1 ml RLT provided in kit.)
3. Mix by pipetting
4. Add 250 µl ethanol to lysate
5. Mix by pipetting
6. Add to spin column and centrifuge at 10,000 rpm for 15 secs
7. Transfer column to new 2 ml tube
8. Add 500 µl RPE buffer
9. Spin at 10,000 for 15 seconds
10. Dump eluant and add another 500 µl RPE
11. Centrifuge on high (14,000 rpm) for 2 min
12. Transfer to new 1.5 ml tube
13. Spin on high (14,000 rpm) for 1 min
14. Transfer column to a new 1.5 ml tube
15. Add 50 µl DEPC water
16. Spin at 10,000 rpm for 1 min
17. Suck up eluant and rerun over column again
18. Spin at 10,000 rpm for 1 min.
(Combine 2 columns into 1=100 µl
Add 2 μl DNase)
Below DNase Treatment
DNase Treatment:
(10 µ buffer)
Combine these things and then incubate at 37˚C for 30 minutes.
Add 20 μl DNase inactivation solution, let sit at room temperature for 5 minutes.
Spin down resin at 14,000 rpm for 1 minute.
Remove RNA supernatant.
Spec at 260 and 280 10 μl + 60 μl H2O]
Want 260/280 ratio to be 1.8-1.9 (~2)
(OD260)(40)(100)(1/1000) = μg RNA per μl. [10μl of gel]