AGAROSE GEL ELECTROPHORESIS FLASHCARDS

Separates DNA fragments by size using an electric current. Negatively charged nucleic acids migrate toward the positively charged electrode, with smaller fragments moving further through the gel matrix.

What is the principle behind agarose gel electrophoresis (AGE)?

Why do larger DNA fragments not travel as far as smaller ones in AGE?

DNA extract samples
Agarose powder
1X TAE buffer
Gel Red
DNA ladder (1kb diluted 1:15)

List at least five essential materials for performing agarose gel electrophoresis.

tracks the DNA migration and helps the DNA sample sink into the well

What is the function of loading dye in AGE?

0.75 g (weigh ~0.76–0.77 g to account for sticking) of agarose powder in 50 mL of 1X TAE buffer

How much agarose powder and buffer are used in preparing the gel?

Overboiling can cause buffer/water evaporation, altering agarose concentration and potentially affecting gel consistency

Why is it important not to overboil the agarose solution?

After cooling the agarose solution for 5–10 minutes but before it begins to solidify.

When is Gel Red added during gel preparation?

creates wells in the gel where DNA samples will be loaded

What is the role of the comb in AGE?

The same buffer (1X TAE) must be used for both preparation and running to maintain consistency in ion concentration and conductivity.

What must you ensure regarding the buffers used for gel preparation and running?

Mix 5 μL of DNA with 1 μL of loading dye = 6 μL total per well

What is the recommended loading volume per sample well?

100 volts, 400 amps, 40 minutes.

What voltage, current, and duration were used in this lab’s AGE?

The cathode (black) is near the wells (top/negative), and the anode (red) is at the bottom (positive); DNA migrates from negative to positive.

What is the proper orientation of electrodes in AGE?

The gel is exposed to UV light using a gel documentation system. Gel Red fluoresces under UV to reveal the DNA bands.

How are DNA fragments visualized after electrophoresis?

To avoid damaging the gel and prevent contamination or inaccurate visualization.

Why must the gel be handled carefully after electrophoresis?

The surface should be wiped with alcohol after gel disposal.

What should be done with the gel tray after imaging?

The DNA molecular weight ladder.

What is loaded in Well #2 during the experiment?

No Template Control (NTC)

It checks for contamination. It should show no DNA bands. Presence of bands indicates error (e.g., primer contamination).

Bands were seen in the NTC due to too much primer being used, indicating contamination or improper setup.

What error occurred during the lab demo for NTC, and why?

Bubbles can distort the wells or hinder uniform DNA migration.

Why must bubbles be removed from the gel mold before solidifying?