DNA Electrophoresis Method

This page will show to set up and run an agarose gel for DNA samples. You should already be familiar with the basic principles of electropohresis, and I will assume that you have already run an SDS-PAGE gel for proteins.

You'll perform DNA electrophoresis several times during the quarter. This page isn't about one particular experiment; it describes the general technique that applies to all your DNA gels. For the details of the specific samples that go on a particular gel, see the lab guide.

DNA electrophoresis apparatus

We'll use the Bio-Rad Mini Sub Cell GT for all our DNA electrophoresis this quarter. This type of gel unit is used in nearly every molecular biology lab; many manufacturers make similar gel units. To set up your gel, you'll need to get all the pieces of the gel apparatus, as shown here.

The gel apparatus we're using for DNA is simpler than the SDS-PAGE apparatus, but you'll have to make your own gel. We'll use agarose gels for DNA.

Getting ready to pour the gel

Set up your gel apparatus. Put the gel tray into the electrophoresis chamber. Note that there are two sets of little slots in the gel tray to hold the comb. The tray goes in as shown, with one set of slots toward the black electrode of the gel box.

The black aluminum pieces are gel casting gates; drop them into the slots in the chambr and they will hold the melted agarose in the gel tray while it cools and solidifies.

The comb should be close to the black electrode. The phosphate groups along the backbone of DNA give it a negative charge; it will migrate toward the positive (red) electrode during electrophoresis. Note that there are two sets of slots where you could put the comb. The middle position is there so you could use two combs in one gel if you wanted. This would double the number of samples you could run on the gel, but you'd only be able to run the samples a short distance down the gel. You won't need to use two combs in this lab.

TBE buffer in a graduated cylinder

The running buffer for DNA electrophoresis is 1x TBE. It contains Tris (a buffer), Borate salt to increase conductivity, and EDTA to chelate cations. Dispense 40 ml TBE from the carboy into a graduated cylinder. (In this lab, it's always 40 ml for DNA gels. You'll vary the amount of agarose you use for different gels, but the volume should always be 40 ml to fit the gel tray.

Pour the TBE into a flask

Pour the TBE into a 125-ml Erlenmeyer flask. Don't use a beaker; the flask reduces evaporation and helps prevent the contents from boiling over in the next step.

Weigh out the agarose

Weigh out the appropriate amount of agarose. For each gel, you need the right amount of agarose to accommodate the sizes of the DNA fragments you're trying to separate; larger DNA fragments require a gel with larger pores (lower agarose percentage), or the bands will smear too much. Smaller DNA fragments reqire a gel with smaller pores (higher agarose percentage); otherwise, the DNA will diffuse out and the bands will be fuzzy.

 

Typical agarose percentages for the Bio 6B lab:

  • Plasmid & lambda DNA: use 0.7% to 0.8% agarose to prevent larger fragments from smearing, but around 1.5% if you want to carefully analyze the smaller fragments of a restriction digest.
  • Small PCR products, 200-300 bp: 2% agarose.

Agarose percentage is in terms of mass/volume; a 1% agarose gel would have 1 gram of agarose per 100 ml buffer, or 0.4 g agarose/40 ml buffer for our gel units.

Pour the agarose into the flask

Pour the agarose powder into the flask with the TBE. Microwave your gel flask for approximately 1 minute. The agarose should melt completely, forming a clear solution with no chunks. Caution: after microwaving, the gel solution may be superheated; when you swirl it, sometimes the hot solution boils over. Use a flask gripper to protect your hand.

It's best to microwave one flask at a time; doing two flasks at once seems to take more than twice as long, and it's possible that one flask will boil over before the other is fully melted.

Pipet the ethidium bromide

Add the ethidium bromide after your agarose is fully melted.

Caution: ethidium bromide is considered potentially hazardous (but see the links at the bottom of this page for more information). If it isn't done already, set up a station for ethidium bromide on the back counter, with the ethidium bromide in its secondary container, a P-20 labeled for ethidium use, a box of tips, and a beaker for waste tips. Do not bring the ethidium bromide bottle to your lab table; everyone should go to the ethidium bromide station to do their pipetting.

Bring your flask to the station on the back bench and add 2 μl of the 10 mg/ml stock solution. Final concentration: (10 mg/ml)(2 μl)=(0.5 μg/ml)(40 ml). Swirl the flask briefly to disperse the ethidium bromide.

Do not microwave an agarose solution containing ethidium bromide; heating the solution will vaporize the ethidium.

Pour the gel

Pour the melted agarose into the gel tray after it has cooled for a couple of minutes. Make sure the comb is positioned properly, and the agarose isn’t leaking through under the dams. The agarose will flow around the comb, creating the wells. Once you've poured it, don't move the gel box until the agarose is completely solid. This could take about 15 minutes.

Remove the comb

Remove the comb. Be sure the gel has solidified completely before you do this. The entire gel should have changed from transparent to translucent, and it shouldn't jiggle when you tap the box. The underside of the gel unit should no longer feel warm. Don't rush this! If you pull out the comb too early, the wells will collapse, making it difficult or impossible to put samples in your wells. You'll have to start over with a new gel.

Hold down the gel tray while carefully pulling the comb up. It may help to pour a little TBE buffer on top of the gel before you do this.

Remove the casting gates

Remove the gel casting gates.

Fill the chamber

Pour 1x TBE running buffer into the electrophoresis chamber, filling both sides until there is just enough TBE to cover the gel. The TBE should fill the wells.

Make sure your wells are properly formed. Position your gel so the red connector is toward you, and look straight down into the wells. You should see cleanly defined rectangular wells. If you don't, it could be because the wells have collapsed.

Always position the gel this way when you get ready to load samples into it. By convention, gel photos are always displayed with the wells at the top, and the wells are numbered from left to right. Positioning the gel this way to load it makes it easier to properly keep track of which sample is in each lane.

Mix each DNA sample with sample buffer before loading it into a well of your gel. In most cases, it's best to use the Parafilm method for this. Cut out a small piece of Parafilm (two squares is plenty) and spread it out with the waxy side up. Gently pipet a 3 μl drop of sample buffer onto the Parafilm for each sample. (The exact amount of sample buffer isn't important; you just need enough to make your samples sink into the wells.)

Mix one sample with sample buffer and load it on the gel before mixing the next sample. That way, you won't be able to confuse one sample with another.

The volume of sample will vary for different experiments, but you can use the same volume of sample buffer (2 or 3 μl) every time.

You can get the whole volume of sample + buffer from the parafilm to the gel in one shot. Suppose your sample volume is 20 μl. You have your pipetter set at 20 μl while you take up the sample and mix it with the sample buffer. Then just press the button a little harder (slightly past the first stop) so you can take up the 23 μl of sample + buffer without resetting the pipetter.

Look at the wells

Load the samples into the wells. Sit down with your elbows braced on the table, and use two hands to stabilize the pipetter. Look straight down into the wells. Don't try to stick the pipet tip into the bottom of the well. Hold the tip at the mouth of the well and slowly squeeze the thumb button; the sample will slowly sink to the bottom of the well.


Loading samples into the wells

The blue sample buffer makes the sample clearly visible as it sinks to the bottom. Don't worry if a little bit of sample ends up on top of the gel; as long as most of the sample is in the well, you'll see it. The small amount of sample on top will just disappear.


Another look at gel loading

Here's another look at gel loading; in this picture, I'm using my left hand to stabilize the pipet tip. Be sure you don't poke the tip into the gel. The agarose is likely to plug the tip so the sample can't come out -- or, in a worst-case scenario, you could poke the tip all the way through the bottom of the gel so the sample leaks out the bottom.

Put the lid on

Run the gel. Put the lid on the box. It will only fit one way, with the red lead connected to the red electrode on the gel. The DNA is negatively charged, so it will run toward the red (positive) electrode. Make sure your gel is oriented correctly within the box (wells toward the black electrode). If the gel is in backward, just lift out the gel tray and turn it around. Plug in the lead wires — the red wire to the red plug on the power supply, black to black. It doesn't matter which of the two red or black plugs you use on the power supply.

Set the range to low and the display switch to volts and turn on the power supply. Using the knob, adjust the voltage to approximately 70 Volts. You should see bubbles forming in your electrophoresis chamber. Set the display switch to milliamps, and it should read approximately 40 mA. The exact amount of current isn't important, but make sure you have some current running through your gel box.

Physics refresher: According to Ohm's law, voltage = current x resistance, or: volts = amps x ohms. When you're running a gel, the voltage is held constant by the power supply, regardless the current. If you turn on the power supply with no gel unit connected, you can set the power supply to 70 V, but the current will be 0 mA. In fact, our power supplies never read 0 mA; if there is nothing connected, the display will read approximately 003 mA. If your power supply reads 003 mA, there is no current running through your gel, and the DNA isn't going anywhere. This could happen if you forgot to add running buffer, for example. It could also happen if one of the wires on the gel unit is broken, which happens occasionally, especially if someone bends the red or black wires where they attach to the lid. Be gentle.

How far to run the gel: Without UV light, you won't be able to see the DNA while the gel runs. You'll generally want to run your DNA gels for about 15-50 minutes. There's no hard rule about how far to run your gel; you just need to run it far enough for the bands to be clearly separated. If you're only expecting one band per lane (as on some of our PCR gels), you don't need to run it far at all. For restriction digests with multiple bands, run it until the first dye front gets about halfway down the gel. Running the gel longer will separate your bands more, but it will also cause the bands to become more faint, and they could disappear completely. If you're not sure whether your gel has run long enough, you can always take it out, look at it on the UV transilluminator (as described below) and put it back to run longer.

Another way to get a quick look is to use a UV flashlight. The plastic lid of the gel unit blocks UV, so you'll have to take it off.

When you think your gel has run far enough, put your gloves and safety glasses back on; it's time to take the gel out and get a picture. Follow these steps to get ready for the photo:

  • Turn off the power supply and unplug the red and black leads.
  • Take the lid off the gel box.
  • Spread a piece of plastic wrap on the table for the gel.
  • Lift the gel tray out of the box and let the buffer drain off the gel, then slide the gel out of its tray onto the plastic wrap.
  • Place the gel & plastic wrap on the UV transilluminator.
  • Dry the edge of the plastic wrap with a paper towel and write your group name and the date on the plastic wrap with a fluorescent highlighter. Make sure the highlighter will be illuminated by the UV light, so it will be fluorescent. Make sure the plastic wrap is dry, or the highlighter will run into your gel and turn it bright yellow.
  • Put down the transparent lid on the transilluminator, turn on the UV light, and take a look at your gel.
Set the camera mode to C

Photograph your gel. Once you've got your gel on the transilluminator and the plastic wrap labeled, don't waste too much time looking at the gel; you'll be able to see it better in the image on the computer. Turn on the camera, and make sure the top dial is set on C mode. Raise the transparent lid on the transilluminator and put the camera on top. Make sure that the camera's hood is all the way down over the UV light. The UV light goes off when you lift the lid, but it comes back on when the magnet on the camera hood activates the switch.

Adjust the exposure

Look at the image of your gel on the camera's screen. Use the camera's zoom lever to zoom in a little, making sure that you include your group name in the photo. Use the left and right buttons to adjust the shutter speed, making the image darker or lighter. For a DNA gel, the background should be fairly dark, with bright and clearly visible bands.

Once you've got the right zoom and exposure, hold down the shutter release button and give the camera some time to focus. It's slow. Once it's focused, push the shutter release button all the way while holding the camera steady. The exposure may be long, and if you move the camera, you could get a blurred picture.

Don't leave the camera on the UV transilluminator longer than necessary. The heat from the UV light will make your gel steamy, and the camera's lens will fog up.

Don't try to use other picture-taking modes, like Program or Manual. That's for suckers. The C mode includes the appropriate settings to help you get a good image. You just need to adjust the exposure.

When you've got your picture, put the camera into review mode (the blue arrow) and connect the usb cable to the lab's laptop. The computer should already be set up to automatically copy your image to the appropriate folder on the computer and delete it from the camera. If you took more than one image, please delete all but one.

Turn off the camera when you're done. If it's left in review mode, it won't turn off by itself, and the batteries will wear down.

When you see that you've got a good picture, throw away your gel in the biohazard trash. Wear gloves and safety glasses for this -- remember, the gel contains ethidium bromide.

Later, the instructor will upload all the images to the lab's flickr site, so you'll be able to view it and download it later. It's important to get everybody's gel picture on flickr, so you can compare your gel to others.

You might also want to take a picture with your phone, but be sure there's a photo on the lab camera.

Cleanup: Leave your gloves and safety glases on; you still need to do a little cleanup after you have your gel photo:

  • Wrap the gel in its plastic wrap and throw it in the biohazard trash.
  • Pour the TBE running buffer down the sink.
  • Rinse the gel unit with deionized water, dry it carefully (watch out for those delicate electrode wires), and put it back together with the gel tray inside.
  • Put the comb and the gel casting gates back in the appropriate box inside the cabinet with the gel unit.

Make sure you return all the parts of the gel unit. If someone fails to put the equipment away properly, you might not be able to run your next gel. Taking care of the equipment is the students' responsibility, and you will be graded on it.

Review

Terms & concepts

  • Agarose
  • Ethidium bromide
  • Sample buffer (also called gel loading buffer)
  • TBE
  • Voltage and current (amps or mA)

Review questions

  1. What determines how much agarose you should use in your gel?
  2. What is the UV transilluminator for?
  3. How could you see your DNA while the gel is running?
  4. Why do you see only DNA on the gel, and not protein?
  5. How do you know which end of the gel to place the comb?
  6. Suppose you turn on your power supply to run the gel and find that the milliamps reading is close to zero. What would you check?
  7. If you want to make a gel with 0.8% agarose, how many grams of agarose should you use?
  8. What does sample buffer do in DNA electrophoresis?
  9. Why do you need to use a buffer like TBE in your gel and in the electrophoresis chamber, instead of just using plain water?

References

DNA electrophoresis techniques & videos

All these videos show the same basic techniques; I'm not sure which one will help you most.

Agarose Gel Electrophoresis from AddGene. Technique page with a video showing how it's done.

Agarose Gel Electrophoresis from Bio-Rad. Good 4-minute look at the entire procedure.

How to make an agarose gel and How to load an agarose gel from Synthetic Biology One.

Ethidium Bromide

The Myth of Ethidium Bromide. Derek Lowe, Science Translational Medicine, 2016.

Heresy about Ethidium Bromide. Rosie Redfield, 2006.

Ethidium Bromide, Wikipedia.

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