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
