Contact: Kristin Searcy
Reagent Preparation
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Transfer the solutions to two separate side arm flasks and degas the solutions for 10 minutes. Filter sterilize the solutions through a 0.45mm 45μm filter. Store the solutions in a brown bottle at 4°C for up to one month.
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- Wet the gray sponge and place it in the front casting slot. Place the sandwich assembly on the sponge with the short plate facing you, and turn the handles of the camshaft so the cams lock the sandwich in place.
- Attach tubing to two separate 30mL syringes. With a separate piece of tubing place the Y-connector on one end of the tubing and attach a syringe needle on the other end. Place, and secure with tape, the tip of needle between the two glass casting plates.
- Make sure the cam wheel of the delivery system is in the vertical position by rotating it counterclockwise.
- Transfer 16mL of the two denaturing solutions to two separate vials. When you are prepared to cast the gel, add 16mL 16μL TEMED and 160mL 160μL 10% ammonium persulfate to each vial and invert to mix. (This step and the following 4 steps need to be completed in a timely fashion to prevent polymerization of the gel while casting.)
- Using a 30mL syringe with the tubing attached, withdraw the LO denaturing gradient solution from the vial, and rid the syringe and tubing of any air bubbles. Place the syringe in the syringe sleeve on the LO density side of the delivery system by inserting the attachment screw on the syringe plunger in the lever groove. Repeat this process with the HI density solution.
- Connect the other end of the tubing from both syringes to the Y-fitting, which should be connected to the needle between the two glass plates.
- Slowly rotate the cam wheel so that the gel solution is delivered to the gel sandwich over a 5 minute interval. Stop casting the gel when the solution is about 1 cm from the top of the small glass plate.
- Immediately rinse the tubing with water so that the gel does not polymerize within the tubing.
- Carefully insert the well comb into the gel, assuring that it is straight. Allow the gel to polymerize for 60 minutes.
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