Difference between revisions of "Gel Electrophorosis"
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(Created page with ''''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 g...') |
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| − | '''Aim''' | + | '''Aim''' |
| + | |||
| + | To be able to view the DNA bands under ultra violet light. | ||
'''Apparatus''' | '''Apparatus''' | ||
| − | + | *beaker | |
| − | + | *pippette | |
| − | + | *casting plate | |
| − | + | *electricity generator | |
| − | + | *comb and comb stand | |
| − | + | *nitrile gloves | |
| − | + | *UV light box | |
'''Material''' | '''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''' | '''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''' | '''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''' | '''Observations''' | ||
| + | |||
| + | (1). | ||
Revision as of 07:10, 21 May 2009
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).
