Phenol

The tRNA extraction described below was adapted from a previous method^{50 (link)}. A pre-culture of the desired strain was incubated overnight at 37°C with shaking at 220 r.p.m. 2 ml of pre-culture was diluted into 50 ml of pre-warmed rich medium (2xYT) and the cells were incubated at 37°C with shaking at 220 r.p.m. for 1 –2h. At OD₆₀₀ = 0.5 –1.0, cells were pelleted (5 min at 5,000 RCF). The cell pellet was resuspended in 800 µl of buffer D (NaOAc 50 mM pH 5, NaCl 150 mM, MgCl₂ 10 mM, EDTA 0.1 mM) and transferred to a 2 ml tube. Cells were pelleted (2 min at 5,000 RCF) and the pellet was resuspended in 450 µl of buffer D. 50 µl of liquefied phenol (90% phenol in water from Sigma Aldrich, cat. No. P9346) was added to the cells and the lysis was performed by head-over-tail rotation (15 r.p.m., 15 min, room temperature). The lysed cells were pelleted (25 min, >20,000 RCF, 4°C). ~500 µl of supernatant containing the RNAs was recovered and transferred to a clean tube. 500 µl of chloroform was added to the solution. The tube was thoroughly vortexed for 1 min until a cloudy emulsion was formed. The emulsion was separated by centrifugation (1 min at >20,000 RCF). The top layer containing the tRNAs was recovered (~480 µl). This solution was either frozen at –20°C or immediately processed with periodate as follows.