This is a protocol for measuring the half-life of mRNA in E. coli, growing at steady state in a chemostat, by Rifampicin transcription arrest.
Contact: Caitlin Conboy
Prepare RNase-diminished bench
- Wipe down thoroughly with EtOH
- Use Rnase Zap wipes to clean bench and pipettes
- Wipe all down with paper towel/ H2O, 2x.
Culture growth conditions
- Continuous culture in chemostat.
Transcription arrest and sample collection
- End chemostat mode:
- Pull feed needle from chamber
- Open rubber lid
- Pull “pre-rif” samples:
- Withdraw 2.6 mL culture
- Dispense two replicates of1 mL aliquots into 0.8 mL of stop solution each on ice. Reserve samples on ice until all time points are collected.
- With remaining 0.6 mL, measure OD600 of a 1:2 dilution of culture in media
- At time zero, add Rifampicin stock (50 mg/mL) to a final concentration of 100 μg/mL. (for 20 mL chemostat volume, add 40 μL stock.)
- For each time point, beginning with time = 2 min:
- Withdraw 2.0 mL culture
- Aliquot and reserve on ice as above
- Sample at 30 second intervals over 2 to 6 min window.
- Continue with RNA extraction protocol.
- RNA per sample can be measured by Northern Blot, real-time RT-PCR or other methods.
There’s considerable variation in the literature in reported working concentrations of Rifampicin used to arrest transcription. Rifampicin isn't soluble in water, so most stock solutions are typically in methanol or DMSO. A desirable working concentration of Rifampicin will be high enough to fully arrest transcription, but low enough to avoid toxicity issues with the solvent:
- Bernstein et al.: Working concentration = 500 μg/mL Rifampicin. (Stock in DMSO.)
- Carrier and Keasling: Working concentration = 250 μg/mL Rifampicin. (Stock in methanol.)
- In Smolke, Carrier, and Keasling: Working concentration = 2 mg/mL Rifampicin. (highest I've seen reported)
- Sean Moore, personal communication: Rifampicin at 500 μg/mL to completely arrest transcription. Typically uses 100 – 250 μg/mL, sufficient for his purposes.