Team:Newcastle/IPTG INduction

{{Team:Newcastle/mainbanner}}

=Aim=

The aim of this protocol  is to prepare slides for the ''Bacillus subtilis'' 168 cells which have ''yneA'' integrated into the chromosome 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 OD<sub>600</sub> by using spectrophotometer and record OD 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 OD<sub>600</sub> 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 take 5ml of the sample from the flask and transfer it to a universal tube and add appropriate amount of the IPTG solution in it. In this way we will have 4 universal tubes for a particular sample having different concentrations of IPTG. 
#Incubate the cells at 37°C on a shaking incubator for 120 minutes and inbetween time, check the OD of the cell culture after every 30 minutes.
#After 120 minutes put the cell cultures out on the bench at room temperature and start preparing the slide.
#For slide preparation, first clean the slide with 100% ethanol.
#Now make 10 ml of 1.2% agarose solution (agarose and water solution).
#Now on the slide add 500µl of the agarose solution and but a long coverslip over it and be careful that all the wells are been filled up by the agarose solution.
#After 5 minutes, remove the coverslip by sliding it horizontally so that we dont disturb the agarose layer.
#Now we take 0.5µl of each sample and load it in each well separately and put the coverslip back on the slide.
#Observe the slide using normal phase contrast or fluorescent microscope. 


{{Team:Newcastle/footer}}