Materials

The Qiagen kit contains enough RNA protect reagent for processing 100 ml of culture.

RNA Isolation

1. Revive and precondition cultures to be tested.
2. Add 100 µl of overnight culture to 10 ml of fresh media (DM100) in 50 ml flask.
3. Growth for approximately 5-6 hrs for REL606/607 or evolved strains or 6-7 hrs for Rif^R ancestors with large fitness defects.
4. Quickly take a small aliquot from each sample for measuring in the spectrophotometer and return cultures to incubator. Measure OD₆₀₀. When harvesting, the OD₆₀₀ should be approximately 0.5-0.6 to get exponential phase RNA. The final OD in this media after 24 hrs of growth reaches approximately 0.16.
5. Take 8 ml of remaining culture. This will contain approximately 5 x 10⁸ cells. Divide equally into two 15 ml conical tubes. Add 2x volume of RNA protect reagent to each.
6. Follow remaining steps of RNeasy Mini kit protocol for processing 5 x 10⁸ cells.

RNA Quality Check

At the CAFG, use the Agilent 2100 Bioanalyzer to check the quality of RNA samples. There are twelve samples per $30 chip. Select the "Bacterial RNA assay option". The ratio of the 16S RNA to 23S RNA peaks should be 1.9 in a high quality sample.

cDNA Synthesis

• Use only random hexamers.
• Save 1/10 reaction before every purification step.

RNA Labeling

RNA Hybridization

Slide Scanning