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now: Thu Jun 24 09:10:58 2021 ... mod: Sun Apr 4 18:41:13 2021
The gel cassette sandwich casters consist of the casting stand, casting frames, and glass plates making sure that these items are clean and dry before setting up the casting stand assembly. Note: At any time, please refer to diagrams in the Mini-Protean 3 Cell Instruction Manual.
1. Place the casting frame upright with the pressure cams in the open position and facing forward on a flat surface.
2. Select a spacer plate of the desired gel thickness and place a short plate on top of it.
3. Orient the spacer plate so that the label is up. Slide the two glass plates into the casting frame, keeping the short plate facing the front of the frame (side with the pressure cams).
4. When the glass plates are in place, engage the pressure cams to secure the glass cassette sandwich in the casting frame. Check that both plates are flush at the bottom. Leaking may occur if the plates are misaligned or oriented incorrectly.
5. Engage the spring loaded lever and place the gel cassette assembly on the gray casting stand gasket. Insure the horizontal ribs on the back of the casting frame are flush against the face of the casting stand and the glass plates are perpendicular to the level surface. The lever pushes the spacer place down against the gray rubber gasket.
6. Repeat steps 1-5 for a second gel.
Pouring the gels
1. Place a comb completely into the assembled gel cassette. Mark the glass plate 1cm below the comb teeth. This the level to which the resolving gel is poured. Remove the comb.
2. Prepare the resolving gel solution with all the reagents except APS and TEMED. Degas the solution under vacuum for at least 15 minutes.
3. Add APS and TEMED to the degassed solution and pour to the mark using a glass or disposable plastic pipette. Pour the solution smoothly to prevent it from mixing with air.
4. Immediately overlay the solution with water or t-amyl alcohol.
5. Allow the gel to polymerize for 45 min to 1 hour. Rinse the gel surface completely with distilled water. Do not leave the alcohol overlay on the gel for more than 1 hour because it will dehydrate the top of the gel.
6. Prepare the stacking gel solution. Combine all reagents except APS and TEMED. Degas under vacuum for at least 15 minutes.
7. Before casting the stacking gel, insert a piece of filter paper to dry the area in between the glass plates above the resolving gel. Take care not to touch the surface of the gel.
8. Add APS and TEMED to the degassed stacking gel solution and pour the solution between the glass plates. Continue to pour until the top of the short plate is reached.
9. Insert the desired comb between the spacers starting at the top of the spacer plate, making sure that the tabs at the ends of each comb are guided between the spacers. Seat the comb in the gel cassette by aligning the comb ridge with the top of the short plate.
10. Allow the stacking gel to polymerize for 30-45 minutes.
11. Gently remove the comb and rinse the wells thoroughly with distilled water or electrophoresis buffer.
12. Rinse the casting frames and stand with distilled, deionized water after use.
1. Remove the gel cassette assemblies from the casting stand. Rotate the cams of the casting frames inward to release the cassette sandwich.
2. Place a gel cassette sandwich into the slots at the bottom of each side of the electrode assembly making sure that the short plate faces inward toward the notches of the U-shaped gaskets.
3. Lift the gel cassette sandwich into place against the green gaskets and slide into the clamping frame.
4. Press down on the electrode assembly while closing the two cam levers of the clamping frame to form the inner chamber and to insure a proper seal of the short plate against the notch on the U-shaped gasket. Short plate must align with the notch in the gasket.
5. Lower the inner chamber assembly into the mini tank. Fill the inner chamber with 125mL of electrophoresis buffer until the level reaches halfway between the tops of the taller and shorter glass plates of the gel cassettes.
6. Add 200mL of electrophoresis buffer to the mini tank, or lower buffer chamber.
1. For sample loading, locate the wells made previously with the various combs.
2. Load the samples into the wells with a pipette using disposable tips being careful not to puncture the bottom of the well with the pipette tip and also slowly enough to allow them to settle evenly on the bottom of the well.
If using color standard lane markers, make sure to load them at this time!
1. Place the lid on the mini tank. Make sure to align the color coded banana plugs and jacks. The correct orientation is made by matching the jacks on the lid with the banana plugs on the electrode assembly. A stop on the lid prevents incorrect orientation.
2. Insert the electrical leads into a suitable power supply with the proper polarity.
3. Apply power to the Mini-Protean 3 cell and begin electrophoresis. 200 volts constant is recommended for SDS-PAGE. Run time is approximately 35 minutes at 200 volts for SDS-PAGE.
1. After electrophoresis is complete, turn off the power supply and disconnect the electrical leads.
2. Remove the tank lid and carefully lift out the inner chamber assembly. Pour off and discard the electrophoresis buffer.
3. Open the cams of the clamping frame. Pull the electrode assembly out of the clamping frame and remove the gel cassette sandwiches.
4. Remove the gels from the gel cassette sandwhich by gently seperating the two plates of the gel cassette. The green, wedgeshaped, plastic gel releaser or a metal spatula may be used to help pry the glass plates apart.
5. Remove the gel by floating it off the glass plate by inverting the gel and plate in a glass dish containing transfer buffer (see WesternBlotting protocol for transfer buffer recipe) agitating gently until the gel seperates from the plate.
6. Rinse the Mini-Protean 3 cell electrode assembly, clamping frame and mini tank with distilled, deionized water after use.