1. Gel Electrophoresis: Introduction and Techniques

2. 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 .

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

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

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

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

7. Making an Agarose Gel

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

9. 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.

10. Electrophoresis Equipment
Power supply
Cover
Gel tank
Electrical leads 
Casting tray
Gel combs

11. Sample Preparation
Loading dye: 
 Bromophenol Blue (for color)

12. 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.

13. Running the Gel

14. Cathode
(-)
 wells
DNA
(-) 
 Bromophenol Blue
Gel
Anode
(+)

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

17. 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