Lab Manual
biological reagents
Cell Free Systems
In Vivo Systems
misc methods

Determination of Transcription Kinetics with DE81 Filter Assay

1. Set up transcription reactions as usual and use 1 ml of 35S-UTP (12.5 mCi/ml;1288 Ci/mmol) per 10 ml reaction.

2. Take 0.5 ml aliquots from the reactions and spot them onto DE81 filters (2.4 cm dia.; Whatman) at 0 (on ice), 5, 10, 20, 30, 60, 90, 120, and 180 min during incubation at 37°C. (Hint: If you are setting up a few reactions, you may want to offset the start of each reaction by 30 seconds or a minute. This will give you time to sample each reaction more accurately. This is especially important at the early time points.)

3. After all the time point samples are collected, wash filters in a beaker containing 0.4M K2HPO4. Use 10 ml per filter and a beaker that is at least twice as large in volume as the volume of K2HPO4 used.

4. Stir the beaker by hand intermittently for 5 min. Repeat this washing step five times.

5. Wash the filters with H2O (10 ml/filter) twice for 5 min.

6. Wash the filters with 95% ethanol once for 2 min.

7. Air dry or use a heat lamp to dry the filters.

8. Put filters into scintillation vials and fill the vials with 5 ml of scintillation fluid (Econofluor).

9. Measure the incorporated radioactivity counts in a scintillation counter.