RNA Extraction with TRIZOL

Wear gloves when handling trizol, work RNase-free!

1. Aspirate medium from cells grown in dishes
2. Wash with 1XPBS
3. In the fume hood, add 1 ml TRIzol per ~1x106 cells, that is when culture flask(25 cm2)reaches confluency; or 4ml of Trizol to cells on plastic of 10cm plate;2.5 ml on 60 mm plate;1 ml on 35mm plate.
4. Pipette up and down for 2-3 min until solution is no longer viscous;
5. Centrifuge for 10 min at 9000 rpm at C( No dissolved protein will be pelleted),and transfer supernatant into fresh tube
6. Add 20% of Chloroform for Trizol ( i.e. 0.2 ml Chloroform for each 1 ml Trizol )
7. Close tubes firmly and vortex vigorously for at least 15 seconds(UP TO 1 min)
8. Let sit at room temperature for 2min 30sec
9. Centrifuge for 15 min at 9000rpm at 4C
10. Transfer upper, aqueous phase to fresh 1.5ml tube(0.5ml/tube), avoid transfer of ANY interface!!
11. Add equal volume isopropanol to the clean supernatant and incubate at RT for 15 min.
12.Centrifuge at 15,000 rpm for 15 minutes to pellet the RNA.
13.Discard the supernatant and resuspend the pellet in 70% ethanol. The RNA can be stored in 70% ethanol at -20°C until use.
14.Prior to use, centrifuge at 15,000 rpm for 15 minutes at 4°C and discard supernatant.
15.Remove the supernatant, and air dry the RNA pellet for 5 min at RT
16.Resuspend the pellet in diethylpyrocarbonate (DEPC) treated water 30ul or RNase-free TE buffer forlabeling.