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Aim

The aim of this protocol is to prepare slides for the Bacillus subtilis 168 cells which have yneA insertion and are induced by IPTG at various concentrations.

Materials

1. Flasks
2. Colonies of Bacillus subtilis 168 cells with different plasmid insertions.
3. Inoculation Loop
4. Shaking incubator
5. 1M IPTG stock solution
6. Appropriate antibiotic solutions
7. 1.2% agarose
8. 12 well glass slide
9. LB broth
10. Spectrophotometer

Protocol

1. Take 20 ml of LB broth with appropriate concentration of antibiotics in a flask and innoculate it with a particular colony of Bacillus subtilis 168 cells with a particular plasmid insertion.
2. Put it onto a shaking incubator at 37°C overnight.
3. Next morning, check the OD600 by using spectrophotometer and record O.D. for future references and to check whether all the cells are in the same growth phase or not.
4. Now,in a flask add 10ml of LB broth with an appropriate concentration of antibiotics and add 100µl of the appropriate overnight culture in it.
5. Measure OD600 after every 30 minutes and record it everytime.
6. In the mean time, prepare flasks with proper labels and also dilute 1M stock solution of IPTG to a range from 0.02mM, 0.2mM, 1mM and 2mM IPTG solutions.
7. When the OD of all the cell population comes to 0.1 then