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4. Sodium dodecyl sulfate (SDS) solution (20% w/v) is prepared by dissolving 20 g SDS in distilled, deionized water to a final volume of 100 ml. 5. Proteinase K solution (20 mg/ml). 6. Saturated NaCl solution (6 M) is prepared by dissolving 36 g sodium chloride in distilled, autoclaved water to a final volume of 100 ml. 7. Pairs of primers which are designed for PCR amplification of ribosomal RNA operon for each bacterial species being studied. 8. Psoralen-PEO-Biotin (PIERCE) is prepared by dissolving it in distilled water to make a stock at 20 mM (about 13.8 mg/ml), stored in aliquots at −20°C, and added to DNA samples as required. The prepared Psoralen-PEO-Biotin solution has to be protected from light by storing in a dark colored tube. 9. UV Cross Linker FB-UVXL-1000 (Fisher Scientific). 10. Sonicator. 11. EPPS–EDTA buffer stored at room temperature: 10 mM EPPS [N-(2-hydroxyethyl) piperazine-N9-(3-propanesulfonic acid)] and 1 mM EDTA (ethylenediamine tetraacetic acid). 2.3. Total RNA Isolation and cDNA Synthesis
1. Liquid nitrogen. 2. Mortar and pestle for crushing the tissues and bacterial cells. 3. Trizol reagent (Invitrogen) for total RNA isolation. 4. Chloroform. 5. DEPC-treated water. 6. Random primers (see Note 1) with a defined sequence at the 5¢ end and random nonamers at the 3¢ end (PCR primer-dN9) are used for both first- and second-strand cDNA synthesis. 7. SuperScript II reverse transcriptase (50 U/ml, Invitrogen). 8. 10× Reverse transcription buffer: 200 mM Tris–HCI pH 8.4, 500 mM KCl, 50 mM MgCl2, and 100 mM DTT. 9. RNase inhibitor (Invitrogen). 10. Klenow fragment (5 U/ml) and 10× Klenow buffer (BioLabs). 11. RNase-free DNase I (Ambion).
2.4. Identification of Bacterial Genes Specifically Expressed Under Desired Conditions
1. Streptavidin-coated magnetic Dynabeads M-280 (Dynal, Lake Success, NY). 2. Magna-Sep Magnetic Particle Separator (Invitrogen). 3. Mineral oil (Bio-Rad). 4. NaCl (1 M). 5. 20× SSC: 3 M NaCl and 0.3 M sodium citrate is prepared as a stock solution and can be diluted to the specified concentration whenever needed.