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rcsA Lysogen Induction
- Grow fresh overnight cultures of lysogens of bacteria carrying the rcsA construct.
- Grow fresh overnight cultures wildtype E. coli.
 Diagram of the induction process. - Make a 1:1000 dilution of lysogen culture and incubate the cultures until they reach an OD600 of 0.1
- More specifically, when the culture is swirled, cloudiness is observed. Check OD on plate reader as it is important to keep OD constant between trials and experiments.
- At this time also make a 1:1000 dilution of the wildtype E. coli culture.
- Add AHL to bring the concentration within the Lysogen culture to 10 nM.
- Resume incubation at 37 degrees C for 1.5 hours.
- At 1.5 hours, add 5% v/v formaldehyde to the lysogen culture and vortex vigorously.
- Spin down cells at 5000xg for 5 minutes.
- Remove an aliquot of the supernatant.
- Follow the phage titering protocols using the supernatant as the phage solution, and titer against the culture of wildtype E. coli.
- Count plaques the following day to estimate the concentration of phage in supernatant.
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