Electroporation of E. coli Cells with Labeled tRNA

E. coli DH5α cells (Invitrogen) were previously diluted five times on 10% glycerol (OD₆₀₀ [optical density at 600 nm] = 60 ± 10) and stored in aliquots of 20 µL at −80 °C. One aliquot of cells (SQ171, for experiments with aminoglycoside-resistant ribosomes) was mixed with 1.5 pmol of Phe-[Cy5]tRNA^Phe or 2 pmol of fMet-[Cy5]tRNA^fMet (prepared as in ref. 28 (link)) and incubated 1 min on ice. The mixture was transferred to a cold (∼4 °C) electroporation cuvette (MBP, 1 mm) and pulsed with 1.9 kV with a MicroPulser (Bio-Rad), which generated a decay time constant of 5.8 ± 0.1. Immediately after, 1 mL of EZ RDM (Teknova) + 0.2% glucose was added to the cuvette to gently resuspend the cells. The suspension was pipetted down to a culture tube and incubated for 30 min at 37 °C and shaking at 200 rpm. The cells were pelleted at RCF 2415 × g for 3 min (MiniSpin, Eppendorf) and washed three times with fresh RDM (room temperature) to remove nonelectroporated tRNA. Cells were finally resuspended in RDM (OD₆₀₀ = 0.02) containing 1 µM SYTOX blue dead cell stain (Invitrogen).