Synthesis of 35SLabeled Riboprobes

1. Warm buffers, nucleotides, DTT, and templates to room temperature.

2. Assemble the following reaction mixture in this order: 10 ||L 35S-UTP, 2 ||L 10X transcription buffer, 1 | L 200 mM DTT, 1 | L RNase inhibitor, 2 | L 2.5 mM NTP, pH 8.O (a mix of CTP, GTP, and ATP to produce 2.5 mM final of each), 1 |L DEPC water. Mix by gently pipeting up and down.

3. Add 1 |L of the appropriate RNA polymerase, and mix gently by pipeting (do not vortex).

4. Add 2 |L (1 |g) of DNA template, mix, and spin for 5 s in microfuge to collect all liquid in the bottom of the tube.

6. Add 10 |L DEPC water, 2 |L yeast RNA, and 2 |L RNase-free DNase, and incubate at 37°C for 15 min.

7. Add 50 | L 5 M ammonium acetate (prepared with DEPC water) and 200 | L ethanol. Incubate on ice for 30 min, and spin in a microfuge for 15 min.

8. Remove supernatant, and immediately dissolve pellet in 30 ||L 10mM DTT.

9. Repeat step 7 of Subheading 3.5.

10. Remove the supernatant, immediately redissolve probe in 20 ||L 100 mM DTT over 15-20 min with occasional vortexing, and count 1 |L in an aqueous scintillant. Calculate probe specific activity in dpm/| L/kb probe length: acceptable probes must be >106 dpm/|L/kb, i.e., specific activity of 1 |L x length of labeled RNA transcript in kb. Store probes at -70°C for up to 2 mo.

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