Cell Lysis Buffer (1 liter)
320 mM Sucrose ------------------------------- 109.6 g
1% Triton X-100 ------------------------------- 20 ml of 50% Triton X-100
5 mM MgCl2 ----------------------------------- 5 ml of 1 M MgCl2
10 mM Tris-HCl (pH7.6) ----------------------- 10 ml of 1 M Tris-HCl
Add deionized H2O to make a final volume of --- 1 liter
Keep at
4oC.
Saline-EDTA (100 ml)
25 mM EDTA (pH8.0) ------------------------- 5 ml of 0.5 M EDTA
75 mM NaCl ----------------------------------- 1.5 ml of 5 M NaCl
Deionized H2O --------------------------------- 93.5 ml
Proteinase K Solution (10 mg/ml)
Proteinase K ------------------------------------ 1 mg
Distilled H2O ------------------------------------ 1 ml
Store at
-20oC.
PROCEDURES
1. Collect animal cells by centrifugation at 1,500g for 10 minutes at 4oC.
2. Resuspend the cell pellet at a concentration of 108 cells/ml of cold Cell Lysis Buffer.
3. Homogenize in a Potter homogenizer with a loose fitting pestle.
4. Centrifuge at 2,500g for 20 min at 4oC to pellet the nuclei.
5. Resuspend the pellet in 8 ml (or, 1/10th volume of Cell Lysis
Buffer from step 2) of Saline-EDTA, and add 0.8 ml of 10% SDS.