Agarose Concentration Range of Separation of

in Gel (% [w/v])

Linear DNA Molecules (kb)

0.3

5-60

0.6

1-20

0.7

0.8-10

0.9

0.5-7

1.2

0.4-6

1.5

0-2-3

2.0

0.1-2

Agarose gels are cast by melting the agarose in the presence of the desired buffer until a clear, transparent solution is achieved. The melted solution is then poured into a mold and allowed to harden. Upon hardening, the agarose forms a matrix, the density of which is determined by the concentration of the agarose.

4. Loosely plug the neck of the Erlenmeyer flask with Kimwipes. If using a glass bottle, make certain the cap is loose. Heat the slurry in a boiling-water bath or a microwave oven until the agarose dissolves. Heat the slurry for the minimum time required to allow all of the grains of agarose to dissolve.

5. Use insulated gloves or tongs to transfer the flask/bottle into a water bath at 55°C. When the molten gel has cooled, add ethidium bromide to a final concentration of 0.5 pg/ml. Mix the gel solution thoroughly by gentle swirling.

IMPORTANT SYBR Gold should not be added to the molten gel solution.

6. While the agarose solution is cooling, choose an appropriate comb for forming the sample slots in the gel. Position the comb 0.5-1.0 mm above the plate so that a complete well is formed when the agarose is added to the mold.

7. Pour the warm agarose solution into the mold.

The gel should be between 3 mm and 5 mm thick. Check that no air bubbles are under or between the teeth of the comb. Air bubbles present in the molten gel can be removed easily by poking them with the corner of a Kimwipe.

8. Allow the gel to set completely (30-45 minutes at room temperature), then pour a small amount of electrophoresis buffer on the top of the gel, and carefully remove the comb. Pour off the electrophoresis buffer and carefully remove the tape. Mount the gel in the electrophoresis tank.

9. Add just enough electrophoresis buffer to cover the gel to a depth of approx. 1 mm.

10. Mix the samples of DNA with 0.20 volume of the desired 6x gel-loading buffer.

The maximum amount of DNA that can be applied to a slot depends on the number of fragments in the sample and their sizes. The minimum amount of DNA that can be detected by photography of ethidium-bromide-stained gels is approx. 2 ng in a 0.5-cm-wide band (the usual width of a slot). More sensitive dyes such as SYBR Gold can detect as little as 20 pg of DNA in a band.

11. Slowly load the sample mixture into the slots of the submerged gel using a disposable micropipette, an automatic micropipettor, or a drawn-out Pasteur pipette or glass capillary tube. Load size standards into slots on both the right and left sides of the gel.

12. Close the lid of the gel tank and attach the electrical leads so that the DNA will migrate toward the positive anode (red lead). Apply a voltage of 1-5 V/cm (measured as the distance between the positive and negative electrodes). If the leads have been attached correctly, bubbles should be generated at the anode and cathode (due to electrolysis), and within a few minutes, the bromophenol blue should migrate from the wells into the body of the gel. Run the gel until the bromophenol blue and xylene cyanol FF have migrated an appropriate distance through the gel.

The presence of ethidium bromide allows the gel to be examined by UV illumination at any stage during electrophoresis. The gel tray may be removed and placed directly on a transilluminator. Alternatively, the gel may be examined using a hand-held source of UV light. In either case, turn off the power supply before examining the gel!

13. When the DNA samples or dyes have migrated a sufficient distance through the gel, turn off the electric current and remove the leads and lid from the gel tank. If ethidium bromide is present in the gel and electrophoresis buffer, examine the gel by UV light and photograph the gel as described in Chapter 5, Protocol 2 . Otherwise, stain the gel by immersing it in electrophoresis buffer or H2O containing ethidium bromide (0.5 Mg/ml) for 30-45 minutes at room temperature or by soaking in a 1:10,000-fold dilution of SYBR Gold stock solution in electrophoresis buffer.

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