Running DNA Gels

Creating 1X TAE

If there is no 1X TAE, we can make it from the 50X TAE that can be found on the chemical shelf. We make this in 2L bottles: 40ml 50X TAE with 1960ml Milliq water (measured using a graduated cylinder). Put into the 2L bottle, screw the cap on, and mix well by shaking the bottle.

Agarose gels

We generally use 0.5% - 2.0% agarose gels; see the image below for more information on which percentage to chose. The agarose is found on the chemical shelf; it is different than the agar used to make plates (it is a more refined version of the same product). We use 100ml 1x TAE for the larger gels or 50ml 1x TAE for a smaller gel. Weigh out the correct amount of agarose (so, 1g in 100ml for a 1% gel) and put in one of the smaller erlenmeyer flasks. Then add the correct amount to TAE and heat in the microwave. Our microwave is powerful — put it on power-level 6 for approximately 2 minutes, but watch it during this time. Do not allow it to boil for more than 3 seconds, as there is then a chance of it overflowing and creating a mess in the microwave. After it boils, use the orange autoclave gloves to take the flask out and gently swish it around. Is the agarose 100% clear and 100% melted? If there are bubbles, are they actively moving up? If not, it needs to be boiled for another 3-5 seconds until all of the agarose is melted.

The gels are cast in the same boxes as we run the gels in; put the tray in perpendicular to how they are run, to create a seal on the open edges of the tray. Then, add the combs to the gel (before the agarose is added). In most cases, it is perfectly OK to use two rows of combs in the gel, especially if you are expecting exactly one band of a predictable size.

We need to wait for the agarose to cool down before casting the gel. Either leave the flask to cool (and occasionally swish it to mix the agarose) or **carefully** use a orange autoclave glove and stream tap water over the flask to cool it. Watch out — if the orange gloves become wet, they will conduct heat! Also, if the agarose cools down too much (if it becomes less than 100% clear, or has bubbles that are not actively rising), then the agarose needs to be re-melted in the microwave. Allow the flask to sit for 5-10 seconds, then test the temperature by touching it without the orange gloves. If it feels very warm, but not burning, then it is ready (~60 degrees).

Add 1ul Ethidium bromide (from the brown bottle in the refrigerator) per 100ml. Swirl the agarose to mix, but try to prevent bubbles from forming. Immediately carefully pour the agarose into the gel cast. Use a pipet tip to either pop any bubbles on the gel (possibly using a pipet to vacuum up bubbles) or move them to the side of the gel. Wait 10 minutes for the gel to cool; by tapping the gel tray, none of the agarose should shake.

Running the gel

The higher the voltage, the faster the gel will run; but, the bands will be blurrier. We run gels between 40V (for publication images) to 80V-100V (for day-to-day gels). Be sure to watch the loading gel and not run your DNA off of the gel!