{"id":134,"date":"2021-11-05T13:04:20","date_gmt":"2021-11-05T12:04:20","guid":{"rendered":"https:\/\/docenciamicrobiologia.umh.es\/?page_id=134"},"modified":"2025-09-21T19:15:00","modified_gmt":"2025-09-21T17:15:00","slug":"extraccion-de-adn","status":"publish","type":"page","link":"https:\/\/docenciamicrobiologia.umh.es\/en\/indice-de-practicas\/7-identificacion-por-biologia-molecular\/extraccion-de-adn\/","title":{"rendered":"Extraction of DNA"},"content":{"rendered":"

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Extraction using the commercial INSTAGENE kit<\/span><\/p>\n

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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\u20105000 rpm for 3\u20105 minutes. The supernatant saline solution is removed and we are left with the cell precipitate. Seal and mark the test tube properly.<\/p>\n

DNA extraction:<\/p>\n

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    1. Add 200 \u03bcL of lysis solution (shake previously) to the precipitate obtained after centrifuging.<\/li>\n
    2. Mix and put it at 56\u00baC for 15 min.<\/li>\n
    3. After this time shake each test tube in the vortex for 10\u201015 seconds.\/li><\/li>\n
    4. Put it in a bath at 100\u00baC for 8 minutes.<\/li>\n
    5. Shake each test tube again for 10\u201015 seconds.<\/li>\n
    6. Centrifuge for 1 minute at maximum speed.<\/li>\n
    7. Use the supernatant (this is where the DNA is located) to carry out the PCR or store at \u201020\u00baC to use it later on.<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n

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      Extraction through temperature cycling<\/span><\/p>\n

      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 \u03bcl of TE buffer is spread over it.<\/p>\n

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        1. \n
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          1. Incubate in a bath at 95\u00baC for 5 minutes<\/li>\n
          2. Incubate in the cold for 1 minute<\/li>\n
          3. Vortex<\/li>\n
          4. Incubate in the cold for 2-4 minutes<\/li>\n
          5. Vortex<\/li>\n
          6. Repeat the whole cycle twice more<\/li>\n
          7. Centrifuge at 9500 rpm for 5 minutes<\/li>\n
          8. Obtain 20 \u03bcl from the top part of the supernatant and transfer it to a new Eppendorf pipette that has been marked correctly.<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n