Ethidium Bromide/Agarose Gels

(Appendix C)

 

Ethidium bromide gels are used for visualization of DNA and RNA. The concentration of agarose depends on the size of the nucleotides. Larger pieces of DNA are run with lower concentrations of agarose, smaller pieces on higher concentration of agarose. For example, DNA or RNA of 250 bp to 12 kb can be run on a 1% agarose gel. DNA or RNA of 500 bp to 25 kb can be run a 0.5% gel. Additionally, higher concentrations of agarose can be used for greater separation of bands. For example, if one DNA sample may have bands at 500 and 550 bp, the individual bands are easier to see if they are run a 2% agarose gel.

 

50x TAE             (1 L)

·       Tris Base                             242 g

·       Glacial Acetic Acid          57.1 ml

·       0.5M EDTA pH 8.0           100 ml

·       Add the above to about 400 ml of H₂O. Mix well and bring to 500 ml with H₂O.

 

 Low Mass Ladder

50 µl ladder

50µl 6X loading dye

200 µl water

 

1.     Set up gel casting tray and make sure the ends of the tray are sealed. Place combs in the tray. Choose combs based on the number and volume of samples.

 

2.     Mix agarose (high melting point) with water. Use 1g per 100ml for a 1% gel, 2g per 100ml for a 2% gel, etc. Leave enough volume to add 50X TAE after dissolving.

 

3.     Heat and stir (or microwave) to dissolve agarose. The solution should be completely clear.

 

4.     Add 50X TAE (2ml for 100ml gel, 4ml for 200ml, etc). Let cool slightly.

 

5.     Add EtBr and mix well(10mg/ml), 10 ul per 100ml agarose/TAE. Remember ethidium bromide is carcinogenic.

 

6.     Pour agarose gel into casting tray and let solidify (gels can be made before needed and stored wrapped in plastic wrap at 4⁰C.

 

7.     Prepare samples for loading. For linearized DNA samples, 1 µl of DNA should be sufficient to visualize. For RNA probe transcripts, 4-7 µl should be sufficient. Bring the volume of the sample up to a larger volume (i.e. 10 µl) with water to make loading easier. For PCR fragments, half or all of the PCR product can be loaded (15-50 µl). No additional water is necessary. Add 6X loading buffer to all samples to dilute to 1X (i.e., 1.7 µl for a total of 10 µl).

 

8.     Place gel into gel apparatus. Cover with enough 1X TAE buffer to fill the apparatus and cover the gel. Attach leads to gel apparatus and power supply (black to black, red to red). Orient the gel such that the wells are closest to the end of the apparatus with the black electrodes (nucleotides are negatively charged and will run toward the positive).

 

9.     Before loading samples, check that apparatus is working properly. Place lid on apparatus. Set to constant voltage. Set power supply to the appropriate voltage for gel apparatus (approximately 80-100 mV for small apparatus, 100-140 mV for large). Push start and verify that bubbles rise from each end of the apparatus.

 

10.  Load samples and start apparatus again.

 

11. Let gel run until the loading dye is approximately 1/2 to 2/3 through the gel.

 

12. Visualize using a UV light box or BioRad gel imager. For the BioRad gel imager, open the Quantity One software. Place gel in light box. In Quantity One, go to File, select Gel Doc. Pus epi white light on light box and orient the gel to center it on the computer monitor image. Close the door and push Trans UV button. Use the controls on the light box to adjust magnification, iris, and focus ad needed. Use the controls on the software to adjust exposure. When ready to take a picture, push “video print” in software.