RNA Extraction using Trizol reagent

[Luke]

RNA Isolation using TRIZOL reagent

1.   Add 1mL Trizol to 1x107 cells, pipette up and down to shear precipitate.
2.   Incubate 5 min at RT or freeze cells at -80 for up to 1 month.
3.   Add 0.2mL chloroform per 1mL TRIZOL.
4.   Shake tubes vigorously by hand for 15s and incubate at RT for 2-3 min.
5.   Centrifuge samples at 12,000g for 15min at 4 degrees.
6.   Layers will form, the bottom being pink organic, the top being colorless aqueous.  Using P200, recover the top layer into new tube. 
7.   Add 0.5mL 2-propanol per 1mL TRIZOL and incubate for 10min at RT.
8.   Centrifuge at 12,000g for 10min at 4 degrees.
9.   Remove supernatant (aspirate).
10.   Wash with 1mL 75% ethanol.
11.   Centrifuge at no more than 7500g for 5 min at 4 degrees.
12.   Aspirate very carefully as the pellet moves easily.
13.   Air dry pellet for 5-15 min.
14.   Dissolve RNA in 20-60uL DEPC-water (RNAse free water).
15.   Measure Absorbance at 260nm.

[V. Balakrishnan]

Slight additions made.