Gel Electrophoresis¶
After PCR, it’s important to confirm that the reaction worked by checking for the presence and size of the product. We do this by running a small portion of each PCR reaction on an analytical agarose gel.
Why We Run a Gel¶
PCR can fail in a variety of ways (no product, wrong size, smearing, etc.). Running a gel lets us visually check:
- Whether a product was formed
- Whether it’s the correct size (~3583 bp for pP6)
- Whether there’s nonspecific amplification
This is an analytical gel, meaning we are only checking—not purifying—the DNA.
What Is a Gel?¶
Agarose gel electrophoresis separates DNA fragments by size. DNA is negatively charged and moves toward the red (positive) electrode when voltage is applied.
- "Run to red": always load your DNA at the black (negative) electrode end.
- DNA travels through a gel matrix—smaller pieces move faster.
- A loading dye is added to weigh down the sample and track progress visually.
Gels are made from agarose, a gelling agent purified from seaweed. To prepare a gel, agarose is dissolved in 1× TAE buffer at 1% weight/volume by heating (typically in a microwave until boiling), then poured into a mold with combs to form wells. After it sets, we store the gels in bulk in a sealed container in the fridge. In lab, you’ll cut a section from a pre-made gel with enough wells for your samples.
TAE stands for Tris-Acetate-EDTA. It is a buffer that maintains pH and ionic strength during electrophoresis. Standard 1× TAE contains 40 mM Tris, 20 mM Acetate, and 1 mM EDTA at pH 8.3.
Lab Sheet Overview¶
Title: TPcon6-P6: Gel (79)
Location of PCR products: Enzyme freezer, PCR rack labeled “to Gel”
Sample Setup¶
You’ll set up one lane for each PCR product. In this case, you're preparing one labeled sample:
| Label | Size | Product |
|---|---|---|
| 79 | 3583 | P6 |
To prepare your gel samples:¶
1) Add 8 µL of loading dye (tube labeled ‘load’) to a new PCR tube.
2) Add 3 µL of PCR product to the tube, mix, and quick spin.
Marker Sample (1 per section):¶
3) Add 8 µL of loading dye to a tube of marker (yellow), mix, and spin.
Running the Gel¶
If others in your lab section are also ready to run a gel, set up one gel for the group:
4) Cut a gel slab with enough wells for all samples plus 1 for marker and a few extras.
5) Place the gel in the rig. Fill with 1× TAE buffer to just cover it.
6) Brace the gel with a plastic wedge to keep it from floating.
7) Label all samples on a strip of paper and arrange tubes in loading order.
8) Using a P20 set to 9 µL, load each sample into a well carefully. We are setting it lower than the full volume to avoid picking up an air gap that can interfere with filling the well. Also avoid puncturing the wells as the liquid will flow out the bottom; if you do, use another lane or start a new gel.
Electrophoresis¶
9) Attach the lid and power leads: "run to red".
10) Run at 175 volts for ~10 minutes, or until the front (blue) dye band is 2/3–3/4 down the gel.
Imaging the Gel¶
11) Transfer the gel to the imager and place a sample label strip above it.
12) Take a picture with your phone through the orange filter.
13) Name the image file by date and time (e.g., 2022_05_23-10am.png) and upload it at: