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:¶

Add 8 µL of loading dye (tube labeled ‘load’) to a new PCR tube.
Add 3 µL of PCR product to the tube, mix, and quick spin.

Marker Sample (1 per section):¶

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. Avoid puncturing the wells—if you do, restart that lane.

Electrophoresis¶

Attach the lid and power leads: "run to red".
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¶

Transfer the gel to the imager and place a sample label strip above it.
Take a picture with your phone through the orange filter.
Name the image file by date and time (e.g., 2022_05_23-10am.png) and upload it to:

Google Drive Upload Folder

🧪 Quiz: Gel Electrophoresis¶

1️⃣ Purpose of Running a Gel

Why do we run an agarose gel after PCR?

To sequence the amplified DNA and analyze the base composition
To purify the PCR product for downstream cloning steps
To amplify the DNA a second time in a gel matrix
To confirm that the PCR reaction worked as expected

2️⃣ Running the Gel

What does "run to red" mean in gel electrophoresis?

Start the gel run using the maximum voltage setting
Load DNA at the red (positive) electrode side
DNA migrates toward the red (positive) electrode
Use red dye to visualize the DNA in the gel

3️⃣ Purpose of Loading Dye

Why is loading dye added to PCR samples before running them on a gel?

To increase DNA yield during electrophoresis
To bind to DNA and make it heavier
To reduce voltage needed for migration
To weigh down the sample and help track it during electrophoresis