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Run-On Transcription
Contributor: The Laboratory of J. Michael Bishop at the University of California, San Francisco
 
Overview
Run-on transcription monitors the regulation of transcription in isolated nuclei.
 
Procedure
A. Preparation of Nuclei - (do everything at 0°C to 4°C in 50 ml tubes)

1. Pellet between 30 and 300 million cells at 1,500 X g for 10 min.

2. Remove the supernatant and resuspend the cells in 20 to 50 ml PBS and repellet the cells at 1,500 X g for 10 min.

3. Remove the supernatant and resuspend the cells in 2 ml RSB and allow cells to swell for 5 min on ice.

4. Add 2 ml RSB/NP-40 and leave on ice for 5 min and check for cell lysis under a microscope.

5. Pellet the nuclei at 2,000 X g for 10 min at 4°C

B. Run-On Transcription Reaction

1. Resuspend the nuclei in 150 2X Transcription Mix and 30 μl 10X Nucleotide Mix, 500 μCi [32P]-rUTP, and bring the final reaction volume to 300 μl with ddH2O (take the volume of the nuclei into account).

2. Incubate at 37°C for 10 min.

3. Lyse the nuclei by adding 1.5 ml of 6M Guanidium Chloride and shear the DNA by passing the sample through a 19 gauge needle on a syringe.

4. Extract the lysate by adding an equal volume of SEVAG and vortexing vigorously. Centrifuge for 30 min at 35,000 rpm at 20°C in a J6M rotor and recover the aqueous phase.

5. Add 2.5 volumes of Ethanol and pellet the nucleic acids by centrifuging at 35,000 rpm at 4°C for 20 min in a J6M rotor.

6. Remove the supernatant and resuspend the pellet in 200 to 500 μl DNase solution and incubate at 37°C for 15 min.

7. Extract the reaction solution by adding an equal volume of SEVAG and vortexing vigorously. Centrifuge for 30 min at 35,000 rpm at 20°C in a J6M rotor and recover the aqueous phase.

8. Add 2.5 volumes of Ethanol and pellet the nucleic acids by centrifuging at 35,000 rpm at 4°C for 20 min in a J6M rotor.

9. Dissolve the pellet in 100 μl 10 mM Tris. Run the solution through a G50 column to separate run-off transcripts from unincorporated nucleotide.

10. Add 2.5 volumes of Ethanol to the purified transcripts and pellet the nucleic acids by centrifuging at 35,000 rpm at 4°C for 20 min in a J6M rotor. Dissolve the transcripts in 250 μl 20 mM HEPES, pH 7.5.

C. Alkaline Hydrolysis of Run-Off Transcripts

1. Add 63 μl of 1 M NaOH to the transcripts and leave on ice for 15 min.