Difference between revisions of "Gel Electrophorosis"

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'''Aim'''  
 
'''Aim'''  
  

Latest revision as of 18:26, 10 December 2010

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. The DNA samples travel ahead through these 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.


Images

<gallery> File:DSC01420.JPG File:DSC01425.JPG File:DSC01456.JPG </gallery>

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.
  • Lastly place the slab of gel on a UV light box and observe.
  • One can also capture a digital image of the same.


Safety measures

  • The chemicals used are highly toxic therefore the use of gloves inevitable.
  • The disposal of tplatic waste used during the process or even chemical should be done in a seperate bin and ultimately to its correct destination (incineration).
  • Washing hands with soap must not be forgotten.


Observations

(1). A ladder is observed as the first column and bands of DNA in the others. (2).DNA markings are observed to have moved from the negative electrode to the positive electrode. (3). The bands are various shades of orange when seen under the UV light since EtBr gets collected between two strands of a DNA. (3). The smaller DNA is seen to move faster towards the positive electrode where as the bigger DNA are slower.


Conclusion

Through the process of Gel Electrophorosis one can observe the DNA bands under UV light and also say that DNA carries negative charge.