Ixon3 897 emccd

DNA and cultures were prepared as previously described⁵⁸ . Briefly, the tRNA/Broccoli fusion sequence was cloned into pET30b between the XbaI and BlpI sites downstream of an inducible T7 promoter. The sequence verified plasmid was transformed into BL21(DE3)-STAR cells (Invitrogen) and single colonies were grown up overnight in Luria Broth (LB) supplemented with 50 µg/mL kanamycin. The overnight culture was used to inoculate fresh LB/kanamycin medium at a 1:1000 dilution and the culture grown at 37 °C to an OD₆₀₀ = 0.4–0.6 before induction with 1 mM IPTG and growth at 37 °C for 2–4 hours. 200 µL of the resultant culture was centrifuged, decanted, and resuspended in 2 mL of M9 minimal salts medium supplemented with 50 µg/mL kanamycin, 5 mM MgSO₄, and 1 mM IPTG. 200 µL of the resuspended culture was transferred to 96-well poly-D-lysine coated glass bottom plates (MatTek) and incubated at 37 °C for one hour. The media was then removed and the wells washed with M9/kanamycin/1 mM IPTG medium before adding 200 µL of M9 media, 1 mM IPTG, and 400 µM DFHBI-1T (Lucerna). The live fluorescence images were taken with an Andor iXon3 897 EMCCD using a 60× oil objective, an excitation filter 472/30, dichroic mirror 490 (long pass) and emission filter 520/40 on a Nikon Ti-E microscope and analyzed with FIJI⁵⁹ .