Extraction of DNA

Extraction using the commercial INSTAGENE kit

Each lab bench has yeast (practical 6) and bacteria to be tested (practical 8). The even number lab benches will use the yeast and the odd number lab benches will use the corresponding bacteria. In both cases, a colony of the microorganism grown in a solid culture medium is selected and it is resuspended in 1 mL of sterile saline solution. It is shaken vigorously until a homogenous suspension is obtained and it is centrifuged at 3000‐5000 rpm for 3‐5 minutes. The supernatant saline solution is removed and we are left with the cell precipitate. Seal and mark the test tube properly.

DNA extraction:

    1. Add 200 μL of lysis solution (shake previously) to the precipitate obtained after centrifuging.
    2. Mix and put it at 56ºC for 15 min.
    3. After this time shake each test tube in the vortex for 10‐15 seconds./li>
    4. Put it in a bath at 100ºC for 8 minutes.
    5. Shake each test tube again for 10‐15 seconds.
    6. Centrifuge for 1 minute at maximum speed.
    7. Use the supernatant (this is where the DNA is located) to carry out the PCR or store at ‐20ºC to use it later on.

 

 

 

 

 

 

Extraction through temperature cycling

An isolated bacterial colony is obtained from a pure culture medium plate. After marking it with an indelible marker pen the colony is pricked with an inoculation loop and an Eppendorf pipette with 100 μl of TE buffer is spread over it.

      1. Incubate in a bath at 95ºC for 5 minutes
      2. Incubate in the cold for 1 minute
      3. Vortex
      4. Incubate in the cold for 2-4 minutes
      5. Vortex
      6. Repeat the whole cycle twice more
      7. Centrifuge at 9500 rpm for 5 minutes
      8. Obtain 20 μl from the top part of the supernatant and transfer it to a new Eppendorf pipette that has been marked correctly.

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