Preparation of total cellular RNA--提取总RNA方法

1) Harvest 1x10⁵ to 10⁸ 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 H₂ O

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 CHCl₃ . Spin 10min at 8k at 4℃.

7) Repeat step 6 if interface is viscous.

8) Extract with 3mls of CHCl₃ (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

14ml 5M NaCl 140mM 20ml 1M Tris pH7.5 (10mM)

750μl 1M MgCl2 1.5mM 5ml 0.5M EDTA

5ml 1M Tris pH 8.6 10mM 6ml 5M NaCl

2.5ml NP40 0.5% 10ml 20% SDS

78ml H₂O 59ml H₂O