Gel Electrophorosis

Aim

To be able to view the DNA bands under ultra violet light.

Apparatus

• beaker
• pippette
• casting plate
• electricity generator
• comb and comb stand
• nitrile gloves
• UV light box

Material

• Agarose
• Buffer solution- TAE(Tris-Cl, Acetic Acid, Ethylene diamene tetra acetic acid)1.0x pH 8
• Ethidium Bromide (5.25 mg/ml in H2O)
• A low molecular weight dye, bromophenol blue
• Antibiotic resitant gene(from ampicilin)

Preperation

• Make a 1% agarose solution in 100ml TAE, for typical DNA fragments. For 3-4 kb solution of DNA a solution as low as 0.7% is used.
• Heat this mixtue to 37 degree C and let it cool.
• Now on wear gloves and add EtBr of 0.5 ug/ml concentration.
• Stir the solution and pour it into the the casting plate.
• Now insert the comb at one side of the gel about 10mm away from the side.
• Once the gel has cooled down and become solid, pull the comb out.
• The holes that remain are the wells.
• Put the gel along with the casting plate in a tank with TAE, EtBr at the same concentration can be added.
• The gel must be completely covered by TAE and placed such that the wells are at the end electrode passing negative charge.

Procedure

• Using a micropippette inject 2.5µl solution with the DNA ladder into the first well (DNA ladder-DNA of previously known lengths) along with the low molecular weight blue dye.
• In the same way now inject the DNA samples mixed with the blue dye into the other wells.
• Now a current is applied, about 100V for 30 minutes.

Observations

(1).