Holmes, Dawn 1-2
TE Sucrose RNA Extraction
- Clean the hood with 10% bleach, ethanol and RNA away
- Use gloves, RNA away, and a lab coat at all times
- Turn on 37oC hot block with water and 70oC water bath
- Pre-heat acidic phenol
- Do all spins at 4oC and keep samples on ice at all times
1. Remove cell pellet in 50mL conical from -20oC
2. Re-suspend cells in 4 ml cool (4oC) TE Sucrose buffer
a. Use 6.7% sucrose in TE pH 8.0.
3. Disperse 1ml into 2ml screw cap tubes
a. From 4ml we’ll have 4 x 2ml tubes
4. Add to each tube:
a. 2ul RNase inhibitor (Ambicon Superase)
i. Only take out when in use
b. 10ul lysozyme (50mg/ml)
c. 3ul proteinase K (20mg/ml)
d. 30ul 10% SDS (RT) mix gently.
5. Incubate 10 minutes at 37oC (hot blot with water)
6. Centrifuge at maximum speed 15 minutes.
7. Recover supernatant to a new tube
8. Add 500ml hot (70oC) acidic phenol (pH 11.5 Ambion)
9. Add 400ml ChCl3: isoamyl alcohol (Sigma)
10. Mix on rotator 10 minutes with the lights off.
11. Centrifuge on high 5 minutes.
12. Recover top aqueous and inter-phase into a new tube
13. Add 500ml heated acidic phenol
14. Add 400ml ChCl3: isoamylalcohol.
15. Mix on rotator 5 minutes with the lights off.
16. Centrifuge on high 5 minutes.
17. Recover the top aqueous phase only
a. Avoid the interphase at this step if it’s possible
18. Add 100ml 5N Ammonium acetate
19. Mix well.
20. Add 20ml glycogen
21. Add 1ml cold (-20oC) isopropanol
22. Vortex briefly to detach the pellet
23. Precipitate RNA for at least 1 hour at -20oC
24. Centrifuge on high 30 minutes
25. Discard liquid immediately.
26. Add 750ml cold (-20oC) 70% ethanol
27. Vortex to detach pellet
28. Centrifuge on high 10 minutes
29. Remove ethanol immediately
30. Remove remaining ethanol by pipetting
Holmes, Dawn 2-2
31. Dry out pellet for about 8 minutes at room temperature
32. Re-suspend in 30ml DEPC water
33. Vortex briefly.
34. Spin for 30 seconds
35. Let sit at room temperature for 15 minutes.
Clean up RNA with Rneasy RNA Cleanup kit (Qiagen):
1. Combine 4 tube into one tube (~120 µl)
2. Add 350 µl RLT buffer with β-mercaptoethanol
a. If not prepared add 10 µl mercaptoethanol to 1 ml RLT buffer
3. Mix by pipetting
4. Add 250 µl ethanol to lysate
5. Mix by pipetting
6. Add to spin column
7. Centrifuge at 10,000 rpm for 15 seconds
8. Transfer column to new 2 ml tube
9. Add 500 µl RPE buffer
10. Spin at 10,000 for 15 seconds
11. Dump eluant
12. Add another 500 µl RPE buffer
13. Centrifuge on high (14,000 rpm) for 2 minutes
14. Transfer to new 1.5 ml tube
15. Spin on high (14,000 rpm) for 1 minute
16. Transfer column to a new 1.5 ml tube
17. Add 50 µl DEPC water
18. Spin at 10,000 rpm for 1 minute
19. Rerun eluant over column
20. Spin at 10,000 rpm for 1 minute
DNase Treatment:
1. Use the DNA-free kit
2. Combine the following:
a. 50 µl RNA (20 µg)
b. 10 µl Buffer
c. 2 µl Enzyme
3. Make up the total reaction volume to 100 µl with DEPC water
4. Mix with P200 up and down
5. Incubate at 37˚C for 30 minutes.
6. Add 20 µl mixed resin
7. Let sit at room temperature for 5 minutes
8. Spin down resin at 14,000 rpm for 1 minutes
9. Remove RNA supernatant.