Biochemical Techniques and Instrumentation

Procedure

1. Pick up a colony of bacteria and inoculate it in a conical flask containing 100 ml autoclaved Luria broth media supplemented with antibiotic (Ampicillin 100 µg/ml) and incubate overnight in a 37°C shaking water bath at 250 rpm.
2. Pour the culture in a 2.0 ml centrifuge tube and centrifuge at 5000 rpm for 20 min.
3. Discard the supernatant and wash the pellet in 1.0 ml double distilled water. Centrifuge the solution at 1000 rpm for 5 min.
4. Discard the supernatant and resuspend the pellet in 250µl cold alkaline solution I (stored at 4°C). Mix properly so that the pellet dissolves.
5. Add 20 mg of lysozyme in the above solution and mix well. Allow it to incubate on ice for 15 min.
6. Add 500 µl of alkaline solution II and mix properly. Mix the solution by flicking.
7. Then, add 250 µl of alkaline solution III and incubate it on ice for 15 min.
8. Centrifuge the sample at 5000 rpm for 10 min and filter the supernatant into an autoclaved 2.0 ml centrifuge tube.
9. Fill the tube with the same amount of ice-cold isopropyl alcohol (stored at-20°C) and incubate in cold (-20°C) for 30 min.
10. Centrifuge the sample at 5000 rpm for 15 min. Discard the supernatant and air dry the pellet.
11. Resuspend the pellet in 500 µl TE buffer.