GEL ELECTROPHORESIS

INTRODUCTION

Gel electrophoresis is a molecular biology technique used to separate and analyze DNA, RNA, or proteins based on their size and charge. It involves applying an electric field to move charged molecules through a gel matrix, typically agarose or polyacrylamide, which acts as a molecular sieve.

DNA molecules are negatively charged due to their phosphate backbone. When placed in an electric field, DNA migrates toward the positive electrode (anode). Smaller DNA fragments move faster through the gel matrix, while larger fragments move more slowly, allowing separation based on size.

Agarose gels are standard for DNA, but polyacrylamide gels are used for smaller fragments (e.g., <500 bp) or proteins due to smaller pore sizes.

Agarose and agaropectin are two polysaccharide components derived from agar, a gelatinous substance extracted from the cell walls of certain red algae (primarily Gelidium and Gracilaria species). Agar is widely used in molecular biology, microbiology, and food industries.

Agarose is a linear polysaccharide and the primary gelling component of agar. It consists of repeating units of D-galactose and 3,6-anhydro-L-galactopyranose linked by glycosidic bonds, forming a neutral or slightly sulfated structure.

Agaropectin is a more complex, heterogeneous polysaccharide. It contains D-galactose, L-galactose, and other sugar residues, with a higher degree of sulfation, methylation, glucuronidation,  or other chemical modifications compared to agarose.

Materials

• Agarose powder

• Electrophoresis buffer (e.g., 50X TAE or TBE)

• DNA samples (with loading dye)

• DNA ladder (molecular weight marker)

• Gel casting tray, comb, and electrophoresis chamber

• Power supply

• Micropipettes and tips

• Microwave or hot plate

• Ethidium bromide (or alternative DNA stain) Caution: Handle with care

• UV transilluminator (or imaging system)

QUEST

You are provided with:

• Six DNA samples

• The staff listed above

• The bottle of beer (optionally)

Tasks:

Procedure

• Prepare your gel:

  1. Measure out the required mass of agarose depending on DNA size — lower % for larger fragments. For example, to prepare 0.8% agarose solution you need to dissolve 0.8 g of agarose powder in 100 mL buffer

  2. Mix agarose with 1X electrophoresis buffer in a flask

  3. Heat in a microwave until fully dissolved (1–2 minutes, swirl gently to avoid boiling over).

  4. Cool the solution slightly (to ~50–60°C)

  5. Add ethidium bromide (e.g., 0.5 µg/mL) and swirl to mix. Use gloves and work in a fume hood if possible

  6. Pour into a gel tray with a comb inserted. Let solidify (~20–30 minutes)

• Set Up the Electrophoresis Chamber

  1. Place the solidified gel (in its tray) into the chamber.

  2. Submerge the gel with 1X buffer, ensuring it’s fully covered but not floating.

  3. Gently remove the comb to reveal sample wells.

• Load the Samples

  1. Add loading dye (6X) to DNA samples to track migration

  2. Pipette 5–20 µL of each sample into separate wells

  3. Load a DNA ladder in the first and in the last wells for reference

• Run the Gel

  1. Connect electrodes: negative (black) at the sample end, positive (red) at the opposite end (DNA moves toward positive, you know)

  2. Set voltage (e.g., 80–120 V) and run for 30–60 minutes, or until the dye front nears the gel’s end

• Visualize Results

  1. Turn off the power and remove the gel from electrophoresis chamber

  2. Place the gel (without its tray) on a UV transilluminator to view DNA bands (wear UV-protective glasses now or dark ones later)

  3. Take a photo and/or analyze the gel

Safety Notes

1. Use gloves when handling ethidium bromide or stained gels (it's carcinogenic)

2. Wear UV-blocking glasses, when working with UV transilluminator

3. Dispose of gels and buffer in accordance with lab waste protocols