11.7. Remove the comb and rinse the glass with tap water to remove with a soft brush all spilled acrylamide from of the glass and well. Mount the gel on the apparatus. Pre-run for approximately 1/2 hour with 1xTBE as running buffer. The 1x TBE buffer in the lower compartment is supplemented with 0.5 M NaAc, creating a salt gradient. This will slow down the small fragments in the lower part of the gel while improving the separation of the larger fragments in the upper part of the gel. This mimics a gradient gel electrophoresis.
11.8. After the pre-run rinse the well from acrylamide rubble and diffused Urea with a beam of liquid (buffer) using a syringe. Place the comb with the teeth downwards (just on top of the gel surface; teeth just for 0.5 mm in the gel).
11.9. To the 10 μl active PCR product add 20 μl formamide dye (98% formamide, 10 mM EDTA, 0.025% bromophenol blue and 0.025% xylene cyanol FF). Denature the samples with the formamide dye by heating them for 5 min. at 92ºC. Place samples on ice and load 2 to 5 μl. Leakage from the end wells can be prevented by loading a counterweight of loading buffer in the adjacent wells.
11.10. Electrophoresis is performed at constant power of 80 Watt per gel to give a constant heat development during running of the samples. The temperature should be 55ºC. After 2.5 hours of running (The Bromophenol Blue is compressing at the bottom or just running off. The Xylene Cyanol is at approx. 10 to 15 cm of the bottom) the run will be stopped and dismount the gel.
11.11. Remove the tape from both sides, and use a wedge to open the glass. The gel is covered by a Whatmann 3MM paper and attached to the paper it is removed from the glass. The other side of the gel is covered with Saran Wrap. Work fast because the Whatmann will swell from water, which will cause rims in the paper during drying on a standard slab dryer at 80℃for two hours. Use several sheets of filter paper to keep the gel dryer support plate free from activity.
11.12. Place the dry gels are placed on X-ray films (Kodak X-omat LS); make marks on the gel and film so that you will be able to align them afterwards, and developed after an appropriate exposure (1-3 days).
12. 2Protocol for the isolation of amplified fragments from poly-acrylamide gels
12.1. Mark the film and the gel to orientate it while in the cassette. Develop the film normally.
12.2. Place the dried film onto the gel, lining up the orientation marks and identify the bands to be isolated (usually done best by placing film and gel on a light box and pencil marking band positions on the Whatman paper. Cut out the band and monitor the activity.
12.3. Incubate in 200 μl TE buffer for 2h at room temperature. Use 5 μl for direct PCR using same primers and conditions as in the pre-amplification.
Alternatively:
12.3. Cut out pieces of Whatman DE81 paper to the size of the gel fragments and insert both into incisions in a 2% agarose gel. Electro-elute at 100 V for 15 min.
12.4. Take out DE81 paper and remove excess TEA buffer on Whatman 3MM (do not dry out). Monitor activity on both gel fragment and DE81 paper - 90% of the activity should now be on the DEAE paper.
12.5. If necessary trim off excess paper and place in a 500 μl tube with a small hole punched in the base. Insert tube into a 1.5 ml eppendorf tube. Spin off remaining TEA buffer and wash twice with 100 μl LSB.
12.6. Elute DNA with 50 μl HSB at R/T for 15 min (for fragments larger that 500 bp, heat to 56°C for 10 min). Spin down supernatant into a new eppendorf tube. Check activity on the remaining paper. If activity is left on the paper, repeat elution.
12.7. Precipitate the DNA with 2.5 volumes of ethanol.
（2 Since fragment isolation from PAGE can give rise to various artefacts, it is important to verify band identity using the appropriate protocols.）
