for Modern Biology© reagents and electrophoresis equipment
Preparation of Electrophoresis Buffer (Tris-Acetate-EDTA, pH 8.0)
To prepare the buffer, add 33 mL of 100X (100 times more concentrated)
buffer solution to 3.3 liters of distilled/deionized water and stir the solution.
This will dilute the buffer to normal strength (1X). Store the buffer in the
refrigerator between electrophoretic runs. The buffer can be reused in the electrophoresis
chamber for at least 3 electrophoretic runs. However, fresh buffer should be
used for preparing the gels.
Preparation of Gel Stain (Methylene Blue)
Prepare the solution by combining 1 mL of the stain concentrate
to 1 liter of distilled/deionized water. The staining reagent is stable for
several months if it is stored tightly capped in the refrigerator.
Preparation of Agarose Gels
1. Place the casting tray on a level work surface and place a precleaned glass slide into the gel support deck.
2. Seal both ends of the gel support deck with tape. The tape must be firmly pressed against the edges of the deck to ensure a tight seal.
3. With the plastic pipette, dispense 15 mL of electrophoresis buffer into a 25 ml glass test tube and add 0.18 grams of agarose. The agarose can be weighed out directly on an appropriate balance. Gently swirl the glass tube until the agarose forms a suspension.
4. Place the test tube into a boiling water bath and allow the agarose suspension to come to a vigorous boil. After boiling for about one minute, remove the test tube from the bath, stir gently with a glass rod, and cool at room temperature for about 2-3 minutes. At this time, the agarose solution should be absolutely clear.
5. Pour the melted agarose directly from the test tube onto the casting deck. Return the test tube to the hot (but not boiling) water bath if you will be loading your gel after casting. The small amount of melted agarose left in the test tube may be used for sample application. Insert the comb into the casting tray slots and push down gently on the top of the comb until resistance is encountered. The teeth of the comb will come to rest in the melted agarose about 0.2 mm above the surface of the glass plate.
6. After the gel has cooled for at least 15 minutes, remove
the tape strips and carefully lift the comb straight up and away from the casting
tray. The gel is now ready for sample application. Gels can also be stored for
up to one week before use. For gel storage, the gel and glass plate is wrapped
in plastic wrap and placed in the refrigerator.
Application of Samples to Gels
1. Hold the micropipetor in a vertical position and place the filling end of the micropipet into the sample solution.
2. Draw the sample into the pipet to the 15 Ál calibration line by lifting up on the handle of the plunger assembly.
3. Wipe excess liquid from the outer pipet surface with a tissue.
4. Carefully direct the filling end of the micropipette into the top of the sample well and slowly eject the 15 Ál of the sample into the well.
5. Draw melted agarose into the micropipet to the 20 Ál calibration line, direct the filling end into the sample well, and slowly eject the agarose onto the sample until the well is full. Between 10-20 Ál of agarose are required to fill the well. The agarose will seal the sample in the sample well.
6. Rinse the pipet by drawing up and expelling water three times from the pipetor.
7. Wipe excess liquid from the outer pipet surface with a tissue.
8. Repeat steps 1-7 to load each additional sample.