dsDNA from the brightest bacterial colonies was PCR amplified from the purified plasmids. Truncation, deletion and point mutation mutants were generated from dsDNAs that were PCR amplified from the appropriate ssDNA templates. Where indicated, dsDNA, and thus the encoded RNA, also contained the tRNA scaffold sequence. PCR products were then purified with PCR purification columns (Qiagen) and in vitro transcribed utilizing an AmpliScribe T7-Flash Transcription Kit (Epicenter). RNA was purified using Bio-Spin columns (Bio-Rad), and quantified using both absorbance values and the Quant-iT RiboGreen RNA Assay Kit (Life Technologies).
Absorption, excitation and emission spectra were measured for solutions using “excess RNA” conditions and limiting amount of fluorophore to ensure that no free fluorophore contributes to the absorbance or fluorescence signal11 (link). The RNA concentration was 20 μM (for the fluorescence measurements) and 50 μM (for the absorbance measurements) while the DFHO concentration was 2 μM and 5 μM respectively.
Absorption, excitation and emission spectra were measured for solutions using “excess RNA” conditions and limiting amount of fluorophore to ensure that no free fluorophore contributes to the absorbance or fluorescence signal11 (link). The RNA concentration was 20 μM (for the fluorescence measurements) and 50 μM (for the absorbance measurements) while the DFHO concentration was 2 μM and 5 μM respectively.
