RNA Precipitation
Note: In general, overnight precipitation gives better yields for all of these protocols.
Isopropanol Precipitation
- Bring up volume to 135 µl and add 15 µl ammonium acetate solution (5M NH4OAc, 100mM EDTA).
- Extract 1X with phenol:chloroform.
- Extract 1X with chloroform.
- Precipitate RNA with 1 volume isopropanol for 30 minutes at -20°C.
- Spin down precipitate 15 minutes at 14,000 rpm at 4°C.
- Wash 1X with 70% ethanol/DEPC
- Resuspend in RNase-free water or formamide.
Lithium Chloride (LiCl) Precipitation
Notes:
- LiCl precipitation does not efficiently precipitate RNA < 300 nts. Use isopropanol precipitation instead
- Efficient LiCl precipitation requires >0.2 µg/µl RNA
- Bring volume up to 50 µl with RNase-free water.
- Add 1/2 volume (25 µl) LiCl solution.
- Incubate >1 hour at -20°C.
- Spin down 15 minutes at 14,000 rpm to pellet RNA.
- Wash pellet with 70% ethanol/DEPC.
- Resuspend RNA in DEPC water (0.1%).
Precipitation from Formamide Resuspension
Note: Suspension in formamide protects the RNA from degradation by RNases.
- RNA can be precipitated from formamide by adding 4 volumes of 100% ethanol at -20°C for 30 minutes to 1 hour.
- Centrifuging at 14,000 rpm for 10 minutes at 4°C.
- Wash pellet with 70% ethanol/DEPC.
- Resuspend RNA in DEPC water (0.1%).