Endy:RNA Half-life

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

 * 1) Wipe down thoroughly with EtOH
 * 2) Use Rnase Zap wipes to clean bench and pipettes
 * 3) Wipe all down with paper towel/ H2O, 2x.

Culture growth conditions

 * Continuous culture in chemostat.

Transcription arrest and sample collection

 * 1) End chemostat mode:
 * 2) *Pull feed needle from chamber
 * 3) *Open rubber lid
 * 4) Pull “pre-rif” samples:
 * 5) *Withdraw 2.6 mL culture
 * 6) *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.
 * 7) *With remaining 0.6 mL, measure OD600 of a 1:2 dilution of culture in media
 * 8) 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.)
 * 9) For each time point, beginning with time = 2 min:
 * 10) *Withdraw 2.0 mL culture
 * 11) *Aliquot and reserve on ice as above
 * 12) Sample at 30 second intervals over 2 to 6 min window.

Sample Processing

 * Continue with RNA extraction protocol.
 * RNA per sample can be measured by Northern Blot, real-time RT-PCR or other methods.