RNA purification/precipitation

From Wikionchus

• This method can be used to:

1. Clean-up RNA after the Trizol extraction
2. Concentrate RNA by precipitation

• All reagents to be prepared in DEPC treated / RNAse free water :'

1. PCI = Phenol:Chloroform: Isoamyl Alcohol solution (25:24:1)
2. 3M NaOAc (pH 5.2)
3. Isopropanol
4. Ethanol 75%

• Method:

1. Add 1/10th volume of 3M NaOAc to RNA solution.
2. Add 2 volumes of PCI (25:24:1)
3. Shake well. Incubate 5 minutes.
4. Spin @ 4C, 14000g, 15 minutes.
5. Take the upper aqueous phase in an new tube.
6. Add max 1ul of Glycogen/Glycoblue per 100ul of solution if needed.
7. Add 2 volumes of Isopropanol. Incubate at -80C for 15 minutes.
8. Spin @ 4Cm 14000g, 15 minutes. Remove suparnatant.
9. Wash pellet with 75% Ethanol.
10. Spin @ 7600g, 4C, 5 minutes.
11. Remove supernatant. Air dry pellet 10 minutes, vacuum dry without spinning for 5 min.
12. Re-suspend pellet in ~25ul of RNAse free water.

• Notes:

1. To visualize very small pellets (< 1ug): Add 0.5ul to 1ul of 15mg/ml Glycogen Blue before adding isopropanol in step 7 above.
2. Sodium Acetate (3M pH5.2) preparation:
   1. NaOAc*3H2O=40.8 gram
   2. Dissolve in 80 ml of ddH2O
   3. Adjust to pH5.2 with glacial acetic acid
   4. Add ddH2O to final volume of 100 ml.
   5. Filter to sterilize.

Links

• Back to Pristionchus pacificus Protocols page