Prepare glass plates for gel:
Rinse plates with cold water, then clean with dishwashing liquid and warm water. Do not scratch plates. Rinse with hot tap water until all soap is removed. The water should form an even sheet over the plate. Rinse thoroughly with tap distilled water, then double distilled water. Rinse with 95% Ethanol and wipe dry with kimwipes, make sure all dust is gone.
Also wash spacers and comb with soap and water. Rinse with distilled water; double distilled water and ethanol.
Put 1.5 mm thick spacers on the 20 x 40 cm glass plate, put the notched glass plate on top of the spacers. Clamp the plates together on one side, top and bottom with large binder clips.
10% Denaturing Acrylamide gel, 100ml (for 20x40 cm glass plates 1.5mm thick)
Swirl and warm up to 42C to allow for rapid polymerization (Heating is optional and not suggested for first-timers )
Pour gel:
Add 678 μl (i.e. if using 100 mls of Urea/Acrylamide) 10% APS (Date:________)
and
67.8 μl TEMED (Lot#___________), swirl to mix and start pouring immediately
with a 50 ml pipette.
Hold the plates at approximately 45 degrees while pouring. Tap the glass to get rid off bubbles. Insert comb and slowly lower the plates down to almost horizontal (put two pipette tip boxes under gel). Pour more gel around comb, wait a few minutes and then try to position the gel horizontal. Leave to polymerize about 1 hour (this depends on the
temperature of the acrylamide, as well as how much temed and APS is added; again I suggest warming the acrylamide up to allow for rapid polymerization). If not running the gel immediately, cover with saran wrap to leave overnight at room temperature.
Pre-running the gel:
Carefully remove comb and spacer on bottom and rinse off any excess acrylamide and urea around comb with syringe. Make sure the wells look clean and no more loose pieces of gel or urea is left.
Stand the gel in the lower buffer reservoir so that the notched glass plate faces the top reservoir (make sure the seal is in it’s groove) and the metal plate in the back. Clamp the gel with the metal plate at the top reservoir on both sides. Make sure the clamp is putting pressure over the spacer between the glass plates and that the clamp is in the
middle of the gasket and not in the middle of the reservoir. The metal plate should not come in contact with the buffer.
Add 1x TBE buffer in both reservoirs. Rinse out the wells with 1x TBE buffer, using a syringe.
Pre run the gel at constant 65 mA until the gel reaches 50℃, about 1 hr. (Glue a temperature strip on the metal plate to monitor the temperature of the gel.)
Load and run the gel:
Mix sample with equal volume 95% Formamide (with BromoPhenol Blue and Xylene cyanol FF).
Heat at 70℃ for 2 min, move to ice.
Turn off power. Rinse out the wells with 1x TBE using a syringe and needle, make sure all urea is rinsed out, load samples.
Run gel at constant 40 mA for about 1 hr (BPB runs around 12nt and cyanol around 55nt.)
