RNA Extraction from Cell

1. Harvest 1x10 5 to 10 8 cells. Wash 1x w / PBS and freeze in liquid N2 and store @ -70 ℃.
2. Resuspend each pellet in 1.5ml lysis buffer (300μl 5x Lysis, 75μl 200mM VR, 1.125ml H2O
   
3. Incubate on ice 5minutes and spin 20min at 8k at 4℃.
   
4. Add equal volume of 2x proteinaseK buffer and 30μl of 20mg/ml ProtK. (Final conc.0.2mg/ml) 
5. Incubate 30 minutes at 42℃
6. Extract with 1-2 volumes of Phenol CHCl 3 . Spin 10min at 8k at 4℃.
7. Repeat step 6 if interface is viscous. 
8. Extract with 3mls of CHCl 3 (Chloroform)  
   
9. Add 2μl of glycogen and 2.5volumes (~7.5ml) of EtOH. PPT at -70℃ or on dry ice. 
10. Spin 10min at 8k at 4℃ in swinging bucket Sorvall. 
11. Wash w/70% EtOH and dry  
    
12. Resuspend in appropriate volume of 1xTE (50-500μl) 
13. Run on a 1% EtBr gel. (28s @ 4.8kb, 18s @ 1.9kb, 5s @ 160bp)

5x Lysis [Final] 2xPK buffer