User:Floriane Briere/Notebook/CHEM-496/2011/09/21

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Objective

We are going to perform an electrophoresis with the DNA with produced yesterday by using the PCR technique. This is going to allow us to determine if the PCR performed well or not. If the some DNA have been produced during the PCR, we'll sequence some DNA molecules to check if they contain the mutation we induced.

Protocol

  • Preparation of the electrophoresis gel
  1. Add 0.25g of Agarose + 25ml of 1X Tris Buffer (buffer is used to favor DNA migration by creating a favorable environment)
  2. Heat for 40 seconds in the microwave
  3. Add TAE buffer (Tris Acetate Electrophoresis buffer; 25mM Tris, 190mM Glycine, 1% SDS)
  4. Leave 25 minutes for the gel to take
  • Run the electrophoresis
  1. In the first weel, we place standard DNA (whose length vary from 500 to 10000bp); it'll be used to identify our DNA (whose length is 3600bp)
  2. Each group place its preparation in a specific weel (5ml of PCR solution + 1ml of a blue solution)
  3. Induce a current through the gel: the DNA is pushed by the negative electrode; the bigger the DNA is, the slower the migration will be.

The blue solution contains some glycerol (to make the DNA going into the gel) and some blue colouring agent (to check that the electrophoresis is running well; and to stop the electrophoresis at the right moment).

  • Revealed the electrophoresis gel: we used Ethidium Bromide to stain the gel.

Results

  • Picture of the electrophoresis gel

Image:21sept - electrophoresis gel.jpg

Conclusion

The left part of the gel corresponds at the DNA standard. We can observe some DNA in the next weel (between the 6th and the 7th standard band). To conclude, we produced DNA during the PCR. The next step is to check if the mutation is contained into our DNA. To do so, we'll sequence our DNA.



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