Before you start the procedure for running a gel, familiarize yourself with the parts of the electrophoresis tank illustrated in "Ward's PAGE System Manual." Associate the diagrams with the actual parts of the apparatus.
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(1)
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With the POSITIVE (red) banana plug to your right as you face the electrophoresis tank, assemble the buffer tanks by inserting the top buffer tank into the bottom buffer tank. The top buffer tank will come to rest on the cassette guide rail bars.
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(2)
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Pour working running buffer solution (1X) into the assembly through the top tank until it reaches "fill line 1."
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(3)
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With the rounded rib side facing you, insert the well forming comb in the thin opening between the two glass plates at the top of the gel cassette. Apply firm but gentle pressure to push the comb between the glass plates. Use the "pusher stick" to press the well-forming comb 1-2 mm into the top surface of the gel.
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(4)
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With the rounded rib side of the well-forming comb facing the front of the tank, insert the cassette assembly into the top tank gasket. Slowly, with a firm, gentle pressure, push downward on the cassette assembly with your thumbs until the assembly seats itself on the bottom of the cassette guide rails in the bottom buffer tank.
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(5)
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Add additional running buffer solution up to "fill line 2." Ensure that the top electrode is immersed in running buffer by temporarily lowering the lid and checking for proper contact level between the running buffer and top electrode.
IMPORTANT:
Do not overfill the buffer compartment.
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(6)
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Use the plastic transfer pipet and the running buffer solution in the system to gently flush any bubbles out of the wells. A strong stream of flow could damage the gel surface and may dislocate the well-forming comb.
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(7)
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Load 10 uL of sample from each microcentrifuge tube onto a corresponding lane. Load 20 uL of the protein marker. If more than one student team is using a gel, keep track of the well number (from left to right) into which a given sample is placed. Demonstrate the correct loading procedure for the students.
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Lane 1 : Yellow Perch
Lane 2 : Walleye
Lane 3 : Chinook salmon
Lane 4 : High molecular weight protein markers
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Teacher's Note:
A 10 uL micropipette is recommended (see "Materials List" for a complete listing of micropipettes). Students should use it to uptake the ENTIRE volume of liquid in the reaction tube. Refer to the "Micromeasurement Section" for directions on usage of micropipettes.
A 13 lane gel cassette can be used by three student teams. Team #1 can load samples into lanes 1-4, team #2 can load lanes 5-8, and team #3 can load lanes 9-12.
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(8)
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Lower the lid assembly onto the electrophoresis tank and snap in the safety lock on the side of the tank.
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(9)
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Make sure the power supply is unplugged and is switched off.
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(10)
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Line up the power supply on the banana plugs: white to red and black to black. Push down firmly on the power supply; you will hear a click when the power supply is properly seated. Note: If you are using a dual tank and running two gels simultaneously, set voltage at 340 V, otherwise set at 170 V. Single voltage power supplies have a preset voltage output.
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(11)
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Insert the 3-prong plug of the power supply cord into a 3-hole 120 volt wall socket. Ensure that the receptacle is properly wired ; i.e. that correct polarity exists.
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(12)
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Depress the power supply on/off button to begin electrophoresis. The button will light up to indicate power is on and electrophoresis is in process. Observe the bubbles that form along the electrodes.
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WARNING:
The power supply must be used only according to the exact instructions provided. Use of this equipment other than as instructed can lead to severe injury or death by electric shock.
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(13)
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Observe the migration of the sample down the gel towards the red (anodic) electrode. Record your observations.
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(14)
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Electrophoresis should continue for 60-75 minutes.
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(15)
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Turn off the power when the loading dye has run half way off the gel. The on/off button light will go out.
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(16)
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After the power has been turned off (on/off button light has gone out), unplug the power supply from the 120 volt wall socket. Then remove the power supply from the banana plugs.
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(17)
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Unlock and open the lid assembly.
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(18)
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The running buffer solution may be hot, so use care in discarding. Pour off enough of the buffer so that the top of the gel cassette is exposed. (The buffer solution can be poured directly down the drain.)
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(19)
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Remove the top buffer tank with the gel cassette(s) still attached.
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(20)
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Remove the gel cassette(s) from the top buffer tank by pulling them down through it. Take care not to change the orientation of the gels (left to right and top to bottom) when handling the cassettes.
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(21)
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Use a scalpel to cut the yellow tape longitudinally along one side of the cassette. CAUTION: Scalpels are sharp; handle with care.
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(22)
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Open the cassette like a book. It may be necessary to insert the edge of the scalpel between the glass plates and gently lever them apart to overcome the adhesion of the spacers. With the scalpel, make a small notch in the lower left corner of the gel. (This will serve as a marker of the gel orientation when you filled the wells with the samples.)
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(23)
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Lift the edge of the gel and slowly place all four fingers of both hands under the high percentage end of the gel (percentage increases from top to bottom). Gently lift the gel and place it in the staining tray.
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(24)
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WEAR PROTECTIVE GLOVES AND GOGGLES. Pour approximately 100 mL of protein stain into the staining tray (just enough to cover the gel). Do not pour stain directly onto the gel. Cover and label. Staining will be complete in about 3 hours. You may stain overnight, but it will take longer to destain.
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(25)
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After staining, carefully decant the used stain. DO NOT allow the gel to move up against a corner. It MUST remain flat; otherwise it could break. Use the "gel handler" to hold the gel down flat during decanting. Decant the stain directly to a sink drain and flush down with water.
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(26)
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Add distilled water to the staining tray. Do not pour water directly onto the gel. The tray may be gently rocked to promote destaining. Destain until bands are distinct, with little background. This will take 6-8 hours, preferably overnight, depending on the degree of agitation used. To enhance the imaging of the bands, you may destain in white vinegar instead of water, or the gel may be stored in white vinegar after it has been destained in water.
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Teacher' s Note:
Use the spatula ("gel handler") provided with this kit to remove the gel from the staining tray. Wrap the gel in clear plastic wrap or use a self-sealing plastic bag and store in a refrigerator.
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(27)
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Wash your hands thoroughly before leaving the laboratory.
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(28)
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Analyze the results.
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