Agarose Electrophoresis

1. Snap the end dams onto each end of the gel casting tray.
2. Insert the gel comb, using either the 8-well or the 16- well side, into the slots near the end of the tray.
3. Melt pre-cooked agarose with a hot bath or a microwave.

-Water bath: Loosen cap on agarose container before placing it in water bath. Water should be at least 100^oC.

-Microwave: Loosen cap on agarose container before microwaving. Heat in one-minute intervals, with swirling, on low power until agarose is melted.

4. After agarose is melted, measure, using a clean graduated cylinder, 25mL of agarose. (figure 1) (technique 1)

-(Optional) Add DNA stain concentrate to the agarose gel to make the DNA bands partially visible as the gel is being run.

-Add 1mL of DNA stain concentrate per 1mL of liquefied agarose- =25mL stain

Add this stain to the liquefied agarose already in the graduated cylinder. Swirl the contents of the cylinder to mix the agarose and stain.

4. Carefully pour agarose into the gel casting tray.
5. Allow the agarose to solidify for approximately 25 minutes. Do not disturb the gel tray or comb. When agarose has solidified, it will turn opaque.
6. Remove the comb and end dams from the casting tray.

Gel Loading

7. Remove 10mL of sample with a clean capillary micropipet. (technique 2)
8. Transfer the sample to a gel well, taking care not to pierce the bottom of the well with the micropipet tip.
9. Repeat the above procedure until all samples have been loaded.

Running a Gel

10. Make sure that the power supply to be used with the electrophoresis chamber, including the power cord and patch cords, are away from any moisture.
11. Place the loaded gel, on the gel tray, in the center of the electrophoresis chamber. Position the well-side of the gel near the negative electrode. (figure 3)
12. Prepare dilute buffer: Add 35mL concentrated 10X TBE running buffer to 315mL R.O. H₂O to obtain a 1X buffer.

-(Optional) Add DNA stain concentrate to the running buffer to make the DNA bands partially visible as the gel is being run.

-Add 1mL of DNA stain concentrate per 1 mL of 1X TBE running buffer.

Add 1X TBE running buffer to one of the side wells of the chamber until the level of buffer is approximately 2mm above the top surface of the gel.

13. Making sure the cover is dry, lock it securely onto the electrophoresis chamber. Wipe off any spills on the apparatus before proceeding to the next step.
14. Making sure that the patch cords attached to the cover, as well as the female plugs and banana jacks on the chamber, are completely dry, connect the red (positive) patch cord to the red electrode terminal on the chamber. Connect the black (negative) patch cord to the black electrode terminal on the chamber.
15. Plug the power cord into a standard 120V outlet.
16. Turn on the power supply. The red light will illuminate, and bubbles will form along the platinum electrodes.
17. Observe the migration of the sample down the gel toward the red (positive) electrode.
18. Turn off the power and unplug the power source when the loading dye has reached the end of the gel. (figure 4)
19. To ensure the utmost safety, wait approximately 10 seconds before disconnecting the patch cords from the power supply.

Staining

20. Carefully push the gel off the gel tray and into the staining tray with a gel handler.
21. Prepare dilute stain: Add 5mL DNA stain concentrate to 95mL warm (50^o to 55^oC) R.O. H₂O.
22. Wearing protective gloves, pour approximately 100mL of warm dilute DNA stain into the staining tray so the stain just covers the gel.
23. Cover and let gel stain for 30 minutes.
24. When the gel has completed staining, carefully decant the used DNA stain directly to a sink drain and flush with H₂O. (technique 3)

Destaining

25. Add warm (50^o to 55^oC) R.O. H₂O to the destaining tray. To accelerate destaining, gently rock the tray. Destain until bands are distinct, with little background color. This will take 30 minutes, depending on the amount of agitation. (technique 4)
26. View the gel against a lighted background such as white paper.

Return to Biology Area

Home Getting Started Admissions & Financial Aid Campus Events Academics Student Life Faculty, Staff, and Administration Community Outreach Campus Directory & Search

For more information, or to report a problem, contact us at webmaster@lima.ohio-state.edu The Ohio State University at Lima, 4240 Campus Drive, Lima, Ohio, 45804 Phone:  419.995.8396 Fax:  419-995-8483 Copyright © 1994-2005, The Ohio State University at Lima. All text and images are the property of The Ohio State University at Lima unless otherwise specified. All rights reserved. Last modified on February 14, 2005.