Team:Newcastle/IPTG INduction
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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
- Flasks
- Colonies of Bacillus subtilis 168 cells with different plasmid insertions.
- Inoculation Loop
- Shaking incubator
- 1M IPTG stock solution
- Appropriate antibiotic solutions
- 1.2% agarose
- 12 well glass slide
- LB broth
- Spectrophotometer
Protocol
- 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.
- Put it onto a shaking incubator at 37°C overnight.
- 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.
- 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.
- Measure OD600 after every 30 minutes and record it everytime.
- 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.
- When the OD of all the cell population comes to 0.1 then
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