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Some of the simple techniques useful for students like me.

1. Overnight 5ml subculture at 37 ℃ for 18hr.
2. Inoculate 100µl subculture into 100 ml LB broth.
3. Culture at 37 ℃, 2½ hr.
4. Centrifuge at 4000 rpm, 10mins at 4°C (50ml +50ml).
5. Re-suspend cell pellet in 5ml 0.1 M cacl2 for each tube.
6. Keep on ice for 5mins.
7. Centrifuge.
8. Finally re-suspend cell pellet in 3 ml 0.1 m Cacl½ (1.5ml for each )
9. Preserve at -70 ℃ by adding 600µl of 50% glycerol ( final 10% gly) and aliquot into 1.5 ml tubes containing 200µl each.