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Team:Caltech/Protocols/rcsA Lysogen Induction
From 2008.igem.org
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| - | + | #Grow fresh overnight cultures of lysogens of bacteria carrying the rcsA construct. | |
| + | #Grow fresh overnight cultures wildtype E. coli. | ||
| + | [[Image:Phage_titer.jpg|right|frame|An example plate. Dark spots are cleared zones in a lawn of bacteria.]] | ||
| + | #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 [[Team:Caltech/Protocols/Titering|<font style="color:#BB4400">titering</font>]] 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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Revision as of 10:14, 23 October 2008
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rcsA Lysogen Induction
 An example plate. Dark spots are cleared zones in a lawn of bacteria.
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