Editing Bacillus sub Expression (section)

==== Making Bacillus competent  ====

#Grow one blank plate of ''Bacillus subtilis'' (or several if you want to transform different strains) for 20 hours at 37ºC (plate been kept on the bench for several days would be better) 
#Inoculate about 12mL of medium with several colonies. Mix the contents of the tube. Check with OD650. Start OD should be between 0.1 and 0.2. Be careful to pipette 0.8mL of this mixture into the cuvette to measure and dispose of it after measurement to avoid contamination in the main mixture. 
#Incubate at 37ºC with vigorous shaking. Read the OD650 every 20min (never keep the solution you used for measuring!) 
#Plot log(OD650) in function of time. After a brief lag, you should observe a exponential increase. After awhile, it will leave the exponential growth; the moment at which it leaves the exponential path is denoted as t0 (3 on the graph). It should take about 100min and the OD should be between 0.35 and 0.55.

OD650 of B.subtilis 

#At t0, incubate for 90 minutes at 37ºC with vigorous shaking. 
#Transfer 0.05mL of this culture into 0.45mL of pre-warmed Medium B in an Eppendorf tube. You have to prepare one tube for each transformation, plus an extra tube for a DNA-less control. 
#Incubate the diluted cultures at 37ºC with shaking for 90min. At this moment, the cells are HIGHLY COMPETENT. 
#To check for competency, you can look at cells under the microscope; competent cells are very motile.