Table 1.

Troubleshooting guide for F30-2xdBroccoli expression and imaging in E. coli

Problem	Possible reason	Solution
No RNA pellet observed after centrifugation (Basic Protocol 1, step 24)	RNA degraded following contamination with RNase Too little cells were taken for RNA isolation	Ensure RNase-free environment. Use only RNase-free solutions and plasticware. Use more cells
Total RNA concentration is too low	RNA degraded following contamination with RNase	See above
No fluorescent bands observed after DFHBI staining	RNA degraded following contamination with RNase F30-2xdBroccoli does not fold when fused to your RNA No F30-2xdBroccoli expression Too weak F30- 2xdBroccoli expression Too short staining time with DFHBI Low concentration of DFHBI in the staining buffer or buffer was used for too many times	See above Test if F30-2xdBroccoli folds and fluoresces when fused to the RNA in study in vitro. Optimize F30- 2xdBroccoli insertion point if needed. Make sure that the strain expresses T7 RNA polymerase Re-make IPTG stock. IPTG, if stored improperly, quickly loses its activity The promoter is too weak. Use T7 promoter. Extend DFHBI staining time Re-make the staining buffer
No distinct individual bands observed after SYBR Gold staining, smear seen in lanes	RNA degraded following contamination with RNase	See above
No fluorescent population observed when using a flow cytometer	Poor RNA-F30- 2xdBroccoli folding in cells Too high threshold setting Incorrect DFHBI concentration Cell incubation time with DFHBI is too short	Optimize F30-2xdBroccoli insertion point Low the threshold Verify that cells were pretreated with 40 μM DFHBI or DFHBI-1T Incubate cell with DFHBI or DFHBI-1T for at least 5 min
Difficulty focusing on cells	Small size of E. coli cells /low cell density Poor magnification of microscope	Focus on cells expressing F30-2xdBroccoli using fluorescence in the FITC channel Make sure the microscope has an objective capable of imaging E. coli (i.e. 60–100X with >1.4 Numeric Aperture)
No adhered cells	Residual LB medium in samples can inhibit cell adhesion No poly-lysine on the dishes	Wash cells with PBS twice before adding cells to dishes Verify that the dishes are pre-coated with poly-lysine
No green fluorescence signal in cells under the microscope	Improper concentration of DFHBI Photobleaching	Check stock solutions and remake imaging media Let the fluorescence to recover in the dark and then re-acquire an image