Gel Electrophorosis
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Revision as of 07:10, 21 May 2009 by 125.17.152.2 (talk)
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).