1. Wash new plates thoroughly using detergent and then dry them completely. Wipe each plate with 100% Ethanol. Siliconize only the plates that have ears.
2. Clamp the plates together, remembering to place a little vacuum grease between the side and bottom spacers, and place a spatula to spread plates slightly at the top to facilitate pouring the gel.
3. To a pouring bottle, add 74 ml of 0.5X TBE Gel Buffer.
4. To a second bottle, add 21 ml of 2.5X TBE Gel Buffer.
5. To the first bottle add 130 μl of 25% Ammonium Persulfate and to the second, add 85 μl of 25% Ammonium Persulfate. Swirl both bottles gently.
6. To the first bottle add 40 μl of TEMED and to the second, add 25 μl of TEMED. Swirl both bottles gently.
7. Bring up 10 ml from the first bottle (0.5X TBE-containing solution) into a pipette and then bring up all of the second bottle's solution (2.5X TBE-containing solution) into the same pipette being careful not to mix the two solutions in the pipette (this is to form a discontinuous gradient).
8. Lift the plates and slowly pipette this solution into the space between them. As it finishes, lower the plate so that a column of gel solution extends up the plate. Pour in the remainder of the first bottle's solution (0.5X TBE-containing solution), maintaining a continuous stream.
9. Push in the flat end of the shark-tooth comb about 4 mm.
10. When gel is set, remove clamps, bottom spacer, and comb. Wash comb area and push teeth of comb into gel. Mark any areas that have bubbles so that you don't load any samples in these regions. Fill tanks with 0.5X TBE buffer.
11. Remove air bubbles where the bottom spacer was using a syringe and needle full of 0.5X TBE buffer. Flush comb out before loading gel.
12. Pre-run gel at 55W (constant power) for 15 min.
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