Oil cfi plan fluor

C. crescentus cells harboring reporter plasmids were serially diluted and grown O/N in liquid PYE/kanamycin medium (5 μg/mL). The next day, cells in the log phase were diluted with fresh liquid PYE/kanamycin (5 μg/ml) to have an OD of 0.05–0.1. The inducer xylose was then added in the medium such that the final concentration of xylose is 0.2%. The cells were grown for 6 h at 28°C at 200 rpm. After this, 2–5 μL of the culture was spotted on M2G 1.5% agarose pads on a glass slide. After the spots soaked into the pad, a coverslip was placed on the pads and the YFP level was measured using fluorescence microscopy using a Nikon eclipse NI-E with CoolSNAP MYO-CCD camera and 100× Oil CFI Plan Fluor (Nikon) objective. Image was captured using Nikon Elements software with a YFP filter cube with exposure times of 30 ms for phase-contrast images and 300 ms for YFP images. The images were then analyzed using a plug-in of software ImageJ (50 (link)) called MicrobeJ (51 (link)).

6 μM of GFP RNase E or GFP RNase E Δ IDR were incubated with 25ng/μL RNase E 5’ UTR - Cy5 RNA in 20mM Tris pH 7.4, 100mM NaCl, 1mM DTT buffer in a total reaction volume of 10 μL for 30 minutes at room temperature. Entire 10 μL was spotted on a slide and covered with coverslip before imaging with Nikon Eclipse NI-E with CoolSNAP MYO-CCD camera and 100x Oil CFI Plan Fluor (Nikon) objective under phase contrast, GFP and Red channels with exposures of 30ms, 50ms and 50ms respectively.