500ml Scale Production of Crude Bacterial Lysate

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1. Place 200ml-2L of superbroth cell culture into a 500ml polypropylene cetrifuge bottle(s). Spin at 2800 x g (Beckman JA-10 rotor) for 10 min. Remove supernatant.
2. Resuspend in 50ml-500ml of Buffer A, spin at 2800 x g (Beckman JA-10 rotor) for 10 min. Remove supernatnat. Repeat.
3. Add 50ml-500ml of lysis buffer, reususpend cells. Leave on ice 30 min.
4. Spin for 1.5hours 200,000 x g (Beckman Ti 50.2 rotor) (45 000 rpm).
5. Place supernatant on ice or freeze at -80ºC.

* add just prior to use