Electrophoresis is a method used with agarose gels to separate different size strands of DNA, consisting of genomic DNA and plasmids, or the products of restriction enzyme digests and PCR. The following is a simple step-by-step procedure for running a gel and visualizing DNA fragments by ethidium bromide staining.
- Choose an Electrophoresis Buffer
Before you prepare the molten gel, you need to decide which electrophoresis buffer to use. Once that is decided, mix the powder agarose with the buffer at the desired concentration, depending on the size range of the DNA strands you are working with. The gel is melted by heating, often in a microwave. The dye for visualizing the separated DNA can also be added at this stage, if you want it within the gel. Alternatively, the gel can be stained by soaking it in dye solution after it is run.
- Pour the gel.
Prepare the gel casting tray by taping or blocking the ends as indicated by the manufacturer. Add the molten agarose to the casting tray and let the gel set. Wait until the gel has cooled to approximately 60 degrees Celsius before pouring, to avoid warping the plastic of the casting tray. Remember to place the sample combs in the gel casting tray prior to pouring the agarose. The combs form wells in the gel into which the samples are deposited prior to running the gel. Allow the gel to cool at room temperature, or, if in a rush, in the refrigerator.
- Prepare the gel apparatus
Remove the sample combs. Carefully remove the combs by pulling straight up out of the gel. Place the gel, still in the casting tray, into the electrophoresis chamber. Remember to remove the end pieces of the casting tray, or tape in some cases, at either end of the gel. Place the casting tray in the gel apparatus such that the end with the wells and the opposite end are in contact with the loading buffer. Also ensure the end with the wells is in line with the cathode, which is usually colored black. The DNA is negatively charged and will migrate towards the positively charged (red) anode.
- Prepare Samples
DNA samples are mixed with loading buffer usually at a ratio of about 1:5 or 1:10. The loading buffer contains a dense substance, usually glycerol, that causes samples to sink to the bottom of the wells, preventing them from diffusing into the running buffer. Tracking dyes present in the loading buffer migrate along with the DNA, ideally with a separation that leaves one migrating behind, and one migrating ahead of the sample. Bromophenol blue and xylene cyanol dyes are often used and migrate at similar rates to double-stranded DNA fragments of 300 and 4000 base-pairs, respectively.
- Load the Gel
Transfer the DNA samples into the wells of the gel using a pipette. Loading DNA samples must be done slowly and smoothly to prevent sample from squirting up into the running buffer and diffusing away.
- Run the Gel
Place the lid on the gel apparatus and connect the leads to a power box. Apply current by turning on the power box and watch for bubbles to form, confirming that a current has been applied. Watch for the tracking dyes to begin migrating in the proper direction (toward the other end of the gel), confirming that the gel has been placed in the chamber with the correct orientation.
Generally gels are run at approximately 80 - 100 mAmp. A setting any higher will cause the buffer to heat up to the point that it begins to melt the gel.
- Monitor the Tracking Dyes
Watch the progress of the tracking dyes and turn off the current before the leading dye has reached the opposite end of the gel.
- Stain the Gel
If dye was not added to the molten gel, stain the gel now in an aqueous solution at concentrations suggested by the supplier. For example, ethidium bromide is used at about 0.5 ug/mL and stained for about 15 to 30 minutes, then destained or rinsed in water, for 10-15 minutes. Some other dyes are available that fluoresce more strongly and might not require destaining. Most are fluorescent dyes that intercalate between the bases of DNA and RNA. Be careful handling them, as this property also makes them potentially very potent carcinogens.
- Visualize the DNA
Place the gel, either alone or in the casting tray, on the transilluminator (UV light of wavelength 254 nm). Remember to wear protective eyewear. Take a photo, if desired, and if you have the equipment. Visualization and photography should be performed shortly after running the gel, as DNA will diffuse over time and the bands will become blurry.
- Load the gel slowly using a small pipette tip, to avoid agitating the running buffer and losing sample from the wells.
- Always wear gloves when handling ethidium bromide and remember protective eyewear when using the transilluminator.
What You Need
- An electrophoresis chamber and power supply.
- Gel casting trays composed of UV-transparent plastic.
- Sample "combs".
- Electrophoresis buffer (usually TAE or TBE).
- Loading buffer containing tracking dyes.
- Ethidium bromide solution.
- Transilluminator (UV light box) and photography equipment (optional).