Gel Electrophorosis

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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