# Agarose gel electrophoresis

[EcoEvoDevo Lab](https://hackmd.io/@EcoEvoDevoLab/AngeliniLab)


Agarose is a polymer that forms a porous gel matrix. DNA fragments of different sizes can be separated by an electrical current that will pull the negatively charged DNA toward the positive electrode. (DNA will “run to the red.”) Smaller DNA fragments will fit through the porous gel more easily, moving farther through the gel than larger fragments in the same amount of time. DNA in the gel is revealed by a fluorescent DNA-binding dye included in the gel.

## Preparation of the gel

Scale this procedure up for larger gels. The concentration of agarose can be varied from 0.8% to 2% for better resolution of larger and small DNA sizes, respectively.

- Set the gel tray in a leveled caster.
- Insert a comb.
- Transfer 50 ml TAE Buffer (1X) into a 250 ml Erlenmeyer flask.
- Add 0.75 g agarose (molecular biology grade) for a 1.5% gel.
- Microwave the flask on high for 66 seconds.
- Afterward, protect your hand from the heat with a rubber mitt or folded paper towel.
- Run the flask under cold tap water for about 10 seconds. The solution should be hot and molten, but not scalding to the touch (50˚C; "baby bottle warm").
- When the solution has cooled, add 6 μl of SybrSafe.
- Swirl gently to mix, but avoid introducing air bubbles.
- Pour the solution into the gel-casting tray. Set the flask aside and let it cool. (See the section below on [agarose waste](https://hackmd.io/@EcoEvoDevoLab/agarosegels#Gel-waste).)
- Remove any bubbles from the gel before it cools. They can be popped or moved to the sides of the gel with a pipette tip. Pay special attention to remove bubbles near the comb.
- Allow the gel to set. When molten, the gel will be clear. Once set, it will be translucent. This takes 10-15 minutes.
- Once the gel has set, remove the comb by pulling it straight up.
- Remove the gel tray from the caster.

:::info
The gel can be used immediately, or it can be placed in a plastic container with a splash of buffer. Close the lid on the container and store the gel at 4˚C overnight.
:::

## Loading the gel

- Place the gel tray in the gel rig.
- The wells must be on the negative (black) side of the rig.
- Add enough buffer (1X) to cover the gel by at least 3 mm.
- Allow any air bubbles in the wells to float out, before loading the gel.
- Cut a strip of ParaFilm about 2 x 10 cm.
- Place ~1 μl droplets of gel loading dye on the ParaFilm strip. One drop for each sample to be loaded. Position them ~1 cm apart.
- One at a time, transfer 6 μl of each sample onto a drop of dye. Mix by pipetting up and down.
- Transfer the dyed sample into a well on the gel.
- Release the sample slowly, and avoid blowing out any bubbles or stabbing the gel.
- Don't forget to add a DNA ladder!
- Once all samples are loaded on the gel, put the lid on the gel rig.
- Connect the leads to the power supply.
- Turn the power supply on, and set the voltage to ~100 V.
- Allow the bromophenol blue dye (purplish-blue) to travel ~5 cm. The farther the DNA runs, the greater ability you'll have to resolve bands of similar size.

:::info
If you can't image the gel immediately when it's run far enough, you can turn the voltage down to 20V for up to an hour.
:::
- When the run is finished, turn off the power supply, and remove the tray carefully from the box. Don’t let the gel slide off the tray!
- Place the gel tray in a plastic container for transport.

## Imaging the gel

:::danger
Never look at a UV light source with unprotected eyes! UV light can kill the cells in your retina, causing permanent blindness. Always wear UV-protective google or a UV-protective face shield if you are working with an exposed UV light source.
:::

:::info
The FotoDyne UV Gel Imager encloses its UV transilluminator. Under normal operation, no proective eye covering is required.
:::


- Carry your gel tray to the FotoDyne UV Gel Imager in Arey 204.
- Check the settings on the device. Be sure that the "Ethidium Bromide Filter" is in place above the light chamber and below the camera.


- Place your gel tray in the center of the chamber, with the wells toward the back.
- Log on to the Mac desktop.
- Open the application ImageJ.
- From the top menu select Plugins/1392 camera/Live.
- On the small white console, press the "trans" button for UV transillumination. An image of your gel should appear in the live window on the computer, although it may be out of focus or off center.


- Adjust the focus and zoom to get the top half of the gel in frame. These settings can be adjusted by turning the 3 rings on the camera. (Top ring is aperture/contrast, middle ring is zoom, and bottom ring is focus). Usually only the zoom ring needs adjustment.


:::warning
If the live image window is too dark, be sure you are fully zoomed out.

You may also need to adjust the software settings. From the 1392 camera plugin window, select `Adjust Settings` from the menu. Turn exposure up to about 1 second. If that doesn’t allow you to see your ladder, turn up the gain.
:::

- If the gel is outside the view area of the camera, adjust the zoom, or open the door to move the gel into the center of the camera's view.
- If it seems like there's really no light coming to the camera, it's possible that the transilluminator has "fail-safed" off. It will do this, for example, if the door is opened while it's on. You can easily reset it by pressing `Reset` on the UV Hi/Lo toggle, which is on the front right of the transilluminator.


- Once the gel is centered and properly exposed, select `Acquire Full Image` from the menu. A static image of your gel will be transferred to ImageJ.
- Invert the image so that it is black bands on a white background. (Humans are better at seeing subtle variation in this direction. And it saves ink when printing.)
- Save the image in jpeg format.
- Print a copy for your lab notebook to the printer next door in Arey 205. (The small thermal paper printer attached to the gel imager is annoying and rarely works.)
- Email a copy of the image file to yourself and/or to Dave.
- Dispose of the used gel in the labeled agarose/SybrSafe container in satellite waste accumulation area in Arey 301.

## Gel image interpretation

When interpreting your gel results, there are several facts to keep in mind. First, be aware that the relationship between a DNA fragment’s size and its migration distance is logarithmic—not linear. So instead of evenly distributed bands in the DNA ladder, smaller fragments separate more (and their bands are more spread out), while larger fragments separate less. This means that the resolution of an agarose gel is better for smaller DNA fragments than for the larger ones. Therefore, your size estimates will be more accurate for smaller fragments. Also beware that if a well is loaded with too much DNA, bands will be more spread out, as lots of co-migrating DNA pushes against its neighboring molecules. The accuracy with which you can estimate the size of these heavy bands is limited. Finally, when you have two different DNA fragments that are close in size, if they are not separated effectively they may appear to be a single fat band instead of two thin bands.

## Gel waste

Agarose and TAE buffer are not toxic. Traditional DNA-binding dyes, such as [ethidium bromide](https://en.wikipedia.org/wiki/Ethidium_bromide), are carcinogenic and terarogenic. For this reason, our lab uses a non-toxic alternative, [SybrSafe](https://en.wikipedia.org/wiki/SYBR_Safe). While far less mutagenic, it is prudent to treat this chemical as a potentially hazardous material. Therefore, we collect it in a waste container labeled agarose/TAE/SybrSafe in the satellite waste accumulation area in Arey 301. This is located to the left and below the microwave.


- Allow the residual agarose gel in flasks used in gel preparation to solidify
- Remove the residual agarose using a chemical scoop or the loop-end of a cleaning brush
- Dispose of the used gels and gel residue in the labeled container in the satellite waste accumulation area
- **Never wash molten agarose down the sink!**

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[EcoEvoDevo Lab](https://hackmd.io/@EcoEvoDevoLab/AngeliniLab)