APPENDIX B

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): agarose cubes q, r, and s containing size-selected DNA (see CHAPTER 13); 1X TAE; 70% ethanol (in a spray bottle); Miracloth squares; BioRad Model 422 Electro-Eluter (cat. no. 165-2976) electroelution system; BioRad PowerPac 1000 power supply (cat. no. 165-5054)

METHODS:

1. Spray down all plastic and glass components of the electroeluter system with 70% ethanol (FIGURE B.1). Rinse these items with distilled water.
2. Pick out three assembly joints and place them on the Parafilm so that the large orifice of each is pointing up. Place a collection cup under each assembly joint and gently fit the collection cup into the small (dorsal) orifice of the assembly joint (FIGURE B.2).
3. Place a 2" x 10" piece of Parafilm on a clean countertop. Place assembly joints, glass tubes, collection cups, and the cuvette rack on top of the Parafilm. Add 1X TAE to each of the three assembly joint/collection cup combinations until a reverse meniscus is visible at the top of each assembly joint. Gently place a piece of sterile Miracloth onto the reverse meniscus of each assembly joint (FIGURE B.2). Insert a glass tube (frosted side down) into each of the three assembly joints. The three resulting structures are referred to as electroelution cuvettes (see FIGURE B.2).
4. Fill each cuvette with 1X TAE. In the first cuvette place one of the three q agarose blocks from CHAPTER 13. Likewise, place an r agarose block in the second cuvette and an s block in the final cuvette. Let agarose blocks drift through the buffer until they come to lie on the Miracloth stretched across the bottom of the glass tubes. If a block does not naturally drift to the Miracloth boundary, gently use a glass rod to push it to this interface. If the cube is too wide to fit in a cuvette, do not force it through the cuvette. Rather, trim the cube until it will fit in the cuvette.
5. Insert each cuvette into a gasket of the cuvette holder as shown in FIGURE B.3. Place gasket plugs in gaskets that are not being used.
6. Place the cuvette holder into the main buffer tank (FIGURE B.4). Add 1X TAE to the main buffer tank until each assembly joint on the electroelution cuvettes is completely submerged.
7. Fill the upper buffer chamber with 1X TAE. Each cuvette should be full of buffer (FIGURE B.4).
8. Place a stir bar in the bottom of the main buffer tank, and set the apparatus on a stir plate. Activate the stir bar so that the buffer in the main tank is gently agitated.
9. Place the lid on the main buffer tank. Make sure that the red (positive) electrode is next to the red dot on the side of the main buffer tank and that the black electrode is next to the black dot on the main buffer tank (FIGURE B.4).
10. Insert the electrode plug at the end of the electrode wires into an appropriate power supply. Make sure that the red electrode is plugged into the red socket and that the black electrode is plugged into the black socket.
11. In general, each cuvette requires 10 mA of power. Thus if three cuvettes are used, the power should be set at 30 mA. If the cuvettes are properly loaded, the voltage should start off at about 80-100 v. Higher voltages indicate possible problems with the cuvettes (e.g., trapped air bubbles). Periodically check the voltage. If voltages > 110 are seen upon starting the apparatus, turn off the power supply, remove the main buffer tank lid, and check to see that the agarose cubes are resting on the Miracloth stretched across the bottom of the glass tubes. Dislodge any bubbles trapped below the agarose cubes or otherwise present in the cuvettes. If the voltage drifts to > 130 v over the course of an hour, decrease the amperage until the voltage is between 80-100 v.
12. Run the electroeluter for 2 hours.
13. Recover electroeluted DNA for q, r, and s blocks as shown in FIGURE B.5. TAKE GREAT CARE IN PERFORMING THIS STEP.
14. Place q, r, and s DNA samples in their own 1.5 ml microcentrifuge tubes on ice.

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