Preparation of DIGLabeled Single Stranded RNA Probe Riboprobe by In Vitro Transcription

1. Linearize DNA with an appropriate restriction enzyme (see Note 2).

2. Extract with phenol/chloroform and precipitate. Resuspend in sterile water at 1 mg/mL.

3. Warm buffers, nucleotides, DTT, and templates to room temperature (see Note 3).

4. Add the following, in the order shown, at room temperature:

b. 4 |L 5X transcription buffer.

j. 1 |L RNA polymerase (T3 or T7). k. x |L (~1 |g) template.

5. Mix gently, not vortex, pulse in microfuge.

7. Check 1 | L on a gel and in the meantime precipitate probe twice as follows: Add 100 |L water, 10 |L 4 M LiCl, 300 |L ethanol, 1 |L glycogen (4 mg/mL). Place at -80°C for at least 1 h. Spin and resuspend pellet in 30 |L of water; add 15 |L 7.8 M ammonium acetate, 1 |L glycogen and 100 |L ethanol. Place at -80°C for at least 1 h. Spin and resuspend pellet in 30 |L of water. Store at -80°C.

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