Trizol extracting RNA from cells
Updated 1/27/2016 12:33 AM
Introduction
Materials
- 1X SDS extraction buffer (Rio)
- 0.5% SDS
- 1mM EDTA
- 20mM Tris pH 7.5
Procedure
- Add trizol directly to dish and homogenize by trituration
Table1
A A | B B | |
1 1 | Dish | Trizol volume (ul) |
2 2 | ||
3 3 | 12 well | 100 |
4 4 | 6 well | 300 |
5 5 | 10cm | 1000 |
- Incubate 5 minutes at RT
- Add 1 volume chloroform per 5 volumes trizol and shake gently (e.g. 0.2mL chloroform per 1mL trizol)
Table2
A A | B B | |
1 1 | Dish | Chloroform volume (ul) |
2 2 | ||
3 3 | 12 well | 20 |
4 4 | 6 well | 66 |
5 5 | 10cm | 200 |
- Rest two minutes at RT
- Spin 10k g for 10 minutes at 4 degrees
- Move the aqueous (clear) phase to a new tube (volume of this should be ~50% total volume from step 4)
- Add 1 volume isopropanol per 2 volumes of the aqueous phase (e.g. 0.5mL isopropanol to 1mL aqueous phase)
- Mix by shaking
- Rest 10 minutes at RT
- Pellet RNa at 10k g for 10 minutes at 4 degrees
- Discard supernatant
- Resuspend RNA in 50µL SDS extraction buffer
- Add 250µL 0.3M NaOAc
- Add 4µL glycoblue
- Add 750µL 100% EtOH
- Put in -80 for overnight.
Modified from life tech and protocol in RNA lab manual by Rio et al