Solution-1 (100 ml)
50 mM Tris-HCl (pH8.0) --------------------- 5 ml of 1 M Tris-HCl
10 mM EDTA -------------------------------- 2 ml of 0.5 M EDTA
10 mg RNase A (heat-treated) ---------------- 1 ml of RNase A (10 mg/ml)
Deionized H2O ------------------------------- 92 ml
Keep at 4oC.
Solution-2 (100 ml)
200 mM NaOH ------------------------------- 4 ml of 5 M NaOH
1% SDS -------------------------------------- 10 ml of 10% SDS
Deionized H2O -------------------------------- 86 ml
Solution-3 (3 M KoAc) (100 ml)
5 M Potassium acetate (pH4.8) ------------------ 49 g
Glacial acetic acid -------------------------------- 23 ml
Add deionized H2O to make a final volume of --- 100 ml
CRITICAL-Adjust pH to 4.8 with glacial acetic acid.
PROCEDURES
1. Pellet 1.5 ml overnight culture of E. coli by centrifugation at 12,000 rpm for 30 second.
2. Remove supernatant. Add 100 ul of Solution-1 to the cell pellet.
3. Vortex to resuspend cells completely.
4. Add 100 ul of Solution-2.
Mix well by inverting tubes. Do not vortex.
5. Incubate tubes at room temperature for 5 minutes.
6. Add 100 ul of Solution-3.
Mix well by inverting tubes. Do not vortex.
7. To precipitate cell debris, centrifuge tubes at 12,000 rpm for
15 minutes.