Gel Electrophoresis Introduction and Techniques Definition Agarose gel

Gel Electrophoresis: Introduction and Techniques

Definition • Agarose gel electrophoresis is a method to separate DNA, or RNA molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). Shorter molecules move faster and migrate farther than longer ones.

What does it do? • Separation of – Proteins – Nucleic Acids • Based on – Charge and/or – Size • What else? – Torture Undergrads

• DNA is negatively charged. • When placed in an electrical field, DNA will migrate toward the positive pole (anode). DNA - Power + • Polymerized agarose is porous, allowing for the movement of DNA

How fast will the DNA migrate? DNA small - Power large +

Agarose D-galactose Agarose is extracted from seaweed. 3, 6 -anhydro L-galactose

Making an Agarose Gel

An agarose gel is prepared by combining agarose powder and a buffer (TAE or TBE) solution. Buffer Flask for boiling Agarose

Staining the Gel • Ethidium bromide binds to DNA and fluoresces under UV light, allowing the visualization of DNA on a Gel. ***CAUTION! Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times.

Electrophoresis Equipment Power supply Cover Gel tank Electrical leads Casting tray Gel combs

Sample Preparation Loading dye: Bromophenol Blue (for color)

Loading the Gel Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.

Running the Gel

Cathode (-) wells Bromophenol Blue DNA (-) Gel Anode (+)

Protocol • For 1% Agarose Gel: • Take 1 gram of Agarose powder in a flask and mix with 100 ml of 1 X TAE or TBE buffer • Place flask in Oven for 3 minutes at Medium temperature • Let the mixture cool down at room temprature

• Add 6 micro liter Ethidium Bromide and pore gel in casting tray • Put Gel combs and let gel to be hard • Place gel in Gel tank • Take 2 micro liter DNA sample and mix with 2 micro liter loading dye and load gel • Run gel for 30 minutes at 100 voltage • Visualize gel in Gel dock