Running DNA Gels

Goal

• Goal: To successfully prepare agarose gel and run DNA gel electrophoresis.

Pre-Protocol Questions

1. Do you have enough 1x TAE?
2. If there is not enough 1x TAE, do you know how to make more?
3. Do you know where the equipment in the lab is? (agarose, flasks, autoclave gloves, Ethidium bromide, etc...)

• Note: check the floor plan for spare gel tanks if needed; there are two complete sets (box+lid with electrodes) for a small gel.

Special Reagents Needed

Creating 1X TAE

• If there is no 1X TAE, make it from the 50X TAE on the chemical shelf.
• Using a graduated cylinder to measure 1960mL MilliQ water, make a 40mL 50X TAE solution.
• Put this solution in a 2L bottle, screw the cap on, and mix well by shaking the bottle.

Agarose gels

Information

• We generally use 0.5% - 2.0% agarose gels; a 'standard' gel is 1% agarose.
• 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 150ml 1x TAE for the larger gels or 100ml 1x TAE for a smaller gel.

1. Weigh out the correct amount of agarose and put in one of the smaller Erlenmeyer flasks.
   • For a 1% gel, 1g in 100ml
2. 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.
3. 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.

See the image below for more information on the absolute best percentage agarose (but notice that a 1% gel is quite good for most purposes).

Casting the Gel

1. Prepare to cast the gel in the same boxes that we run the gel in: Put the tray perpendicular to how they are run, to create a seal on the open edges of the tray.
2. Before agarose is added, add the combs to the gel.
   • 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.
3. Wait for the agarose to cool down before casting the gel.
   • Either leave the flask to cool, occasionally swishing it to mix the agarose, or carefully use an 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).
4. Add 3 uL Ethidium bromide (from the brown bottle in the refrigerator) per 100ml.
5. Swirl the agarose to mix, but try to prevent bubbles from forming.
6. Immediately carefully pour the agarose into the gel cast.
7. Use a pipet tip to either pop any bubbles on the gel. You could use a pipet to vacuum up bubbles or move them to the side of the gel.
8. Wait 10 minutes for the gel to cool
   • To know if it cools completely, tap the gel tray. None of the agarose should shake.

Loading the gel

Switch the gel tray so that we can run the gel parallel to the electric current. Fill the gel tank up to the fill line with 1x TAE, ensuring that the entire gel is submerged. Then, carefully remove the comb by pulling upwards evenly across the comb. The side of the gel with the comb should be at the negative lead, so that the DNA moves towards the positive lead.

Depending on the size of the DNA, load 3 uL of either the 100 bp or 1 kb DNA ladder into the first well.

• Add 6x loading dye to the entire sample that you would like to run. As an example, if you have a 50ul PCR reaction and want to run a fraction of it to ensure that the PCR worked:

In a 0.6ml microcentrifuge tube, add:

• • 2ul of your PCR
  • 8ul of TAE (can be from the gel tank itself)
  • 2ul of 6x Purple Loading Dye (in the refrigerator)

This creates a 12ul solution, with 2ul being dye; this makes the dye at a 1x concentration from 6x. Vortex to mix.

Pipet the entire contents and hold it over the well, but under the water. Slowly push the pipet button to release the mix. The pipet tip should never actually enter the well itself, but it should hover above the well and drop in the contents.

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!