Culture Growth (B. subtilis) 1. Grow an overnight 5.0 mL LB culture of the strains to be evaluated. 2. Inoculate the overnights to 30 mL LB and do a short growth curve to get the cells at mid log-phase (OD600 = 0.5). 3. Pellet cells at 5000 × g for 5 min at 4 °C. All steps and solutions from here forward are to be on ice. 4. Re-suspend pellet in 300 mL cold Sodium Acetate (0.3M, pH 4.5), 10 mM EDTA. 5. Add cells to 0.3 g of glass beads and snap freeze in ethanol and dry ice bath, then transfer to –20 °C. Hold samples here until all cultures are at this point. tRNA Extraction 6. Add 300 mL phenol:chloroform (pH 4.7) to the samples. 7. Vortex eppendorf tubes 4-5 times for 30-second bursts with incubation on ice in between. 8. Centrifuge samples for 15 minutes at 4 °C, at maximum speed. 9. Transfer the top layer (aqueous phase) to new tubes and repeat the phenol extraction by adding 300 µL phenol:chloroform (pH 4.7), spinning for 15 minutes at 4 °C, at maximum speed, and removing the aqueous phase to a fresh eppendorf tube. 10. Precipitate the RNA by adding 3 volumes of cold 100% ethanol and spinning at maximum speed for 25 minutes at 4 °C. 11. Re-suspend the RNA pellet in 60 µL cold sodium acetate (0.3 M, pH 4.5). 12. Precipitate the RNA by adding 400 µL 100% ethanol and spinning at maximum speed for 25 minutes at 4 °C. Decant and spin again for 1 minute, then remove traces of ethanol with a pipette. 13. Air dry the pellet while on ice. 14. Re-suspend the pellet in 50 µL cold sodium acetate (10 mM, pH 4.5). 15. Quantitate RNA by spectrophotometry (A260) and analyze the integrity of the RNA by running a 2.0% agarose gel.