- Using a restriction map, decide on 4-8 restriction enzymes that give 5-12 bands of distinct MW for your DNA. Use these enzymes to cut your DNA, then end-label the DNA using any standard procedure (50-100uCi), then dissolve the final labeled DNA in 250ul ddH2O.
For each labeled DNA:
- Boil 5 (one is a spare) small (10-15mm) nitrocellulose filter circles for 10 minutes, and allow to cool while continuing with the experiment.
- Prepare 4 eppendorf tubes with 2ul labeled DNA and 100ul 1X FBB. Add 0, 0.2, 1, and 5ul RNA polymerase to tubes 1-4 respectively, and incubate at 30C (or another temperature, depending on the RNA polymerase) for 5 minutes.
- Add 5ul NTP mix, and continue the incubation for another 5 minutes, then transfer to ice.
- GENTLY and SLOWLY filter each sample drop-wise through a boiled nitrocellulose filter, then wash the filter by filtering 500ul ice-cold 1X FBB.
- Place the filter in a scintillation vial containing 400ul elution buffer and incubate 1 hour at 37C with shaking.
- Collect the elute from the scintillation vial and add 50ul 2.5M NaOAc. To both the filtrate and eluate, add 400ul phenol:chloroform, vortex, spin, and collect the upper (aqueous) phase. To each, add 1ml EtOH, freeze 5 min in a dry-ice:EtOH bath, then spin 5 min. Discard the supernatant, and dry the pellet in a roto-vac.
- Redissolve the pellets in 20ul each 2X tracking dye, and run on a 5% PAGE (or other gel, depending on the DNA fragment sizes). Dry the gel and autoradiograph overnight with screens (-70C).
If desired, or required, scan the gel lanes with GELSCAN or another densitometer, and quantitate the intensities of each band. The results can then be analysed by hand (don't forget to correct for differences in labeling!) or by the FILTER program.
Usually, eluates from complexes with the lowest amounts of RNA polymerase show the highest specificity and the lowest % retention.