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Natural Science Forum / Biology / Biology / March 2006Osmotic shock - solvent vs culture volume?
Hi everyone. Have a question about standard procedure of osmotic shock. I grow my E.coli after induction by means of IPTG for 12-16h in 27Cdeg. Than I want to make osmotic shock. Many people say many things about proportions of Tris, sucrose, EDTA solution vs volume of culture after harvesting by cetrifugation. For me the lower volumes the better situation for next steps (eg batch with Ni-NTA resign). I read about 80mL of Tris, sucrose, EDTA solution for 1g of culture after spin but also 8mL of this solution for 100mL of culture volume befor spin. What do you advise me? JC >Hi everyone. > [quoted text clipped - 7 lines] >also 8mL of this solution for 100mL of culture volume befor spin. What >do you advise me? Try it and see what works for you. Osmotic shock is a bit tricky; depends on exact conditions, and also on rate of mixing. Further, your constraints may be different from someone elses. So try it, using the lit info as guides, and optimize it for yourself. bob i see ur point Bob. However lit info are in fact at variance with each other, I hope some of u tell me about ur experience. The lit info are in fact not at variance with eachother. 80ml for 1g culture after spin, incidateing 1g of 'cells'. vs 8ml for 100ml of culture BEFORE spin, you won't get 1g of cells out of 100ml of culture. The two values are actually very close. >i see ur point Bob. However lit info are in fact at variance with each >other, So ? You're missing the point. You need to work out what works for you -- your cells, your conditions, your needs. Use the lit as a guide, and make it work for you. bob |
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