21 May 2011

Lima Bean Bacteria DNA Extraction


Materials

    Dry lima beans
    Palmolive detergent
    Centrifuge
    Distilled water
    Centrifuge tube
    Fresh papaya juice
    Graduated cylinder (10ml)
    Non-iodized salt
    Granulated sugar
    Pipet
    Epsom salts
    15 ml test tube
    Bufferin (325mg)
    Test tube rack or 250 ml beaker
    Ice cold 95% ethanol
    Glass stirring rod

Solutions
Lima Bean Bacteria Suspension: Place 1-2 handfuls of dry lima beans in a large jar and fill halfway to the top with distilled water. Cover and sit in a warm room for 2-3 days. Culturing longer than three days often results in more DNA but it usually shears. Pour through a strainer and keep the liquid for the extractions.

Prep buffer solution:

    57 g granulated sugar
    3 g epsom salts
    1 buffered aspirin
    Add distilled water for a total volume of 500 ml

50% detergent solution:

    20 ml detergent
    20 ml distilled water

Salt solution:

    29.2 g non-iodized salt
    Add distilled water for a total volume of 250 ml

Protocol

    Add 14 ml of the bacterial suspension to a centrifuge tube and spin in a balanced centrifuge for 5 minutes.

    Pour off the liquid (supernatant) and discard. You want to keep the pellet as this has your cells.

    Add 5 ml of prep buffer and resuspend your cells with a pipet.

    Add 1 ml 50% detergent solution.

    Add 1 ml papaya juice.

    Add 2 ml salt solution and shake for 2 minutes.

    Place the tube in the centrifuge and spin for 5 minutes. Make sure the centrifuge is balanced.

    Draw off 7 ml of the supernatant (liquid) as this has the DNA and place it in a clean test tube.

    Pour 7 ml of ice cold ethanol carefully down the side of the tube.

    Let the mixture sit undisturbed 2-3 minutes until the bubbling stops.

    The DNA will float in the alcohol. Swirl a glass rod at the interface of the two layers. You may see some tiny threads of DNA but are more likely to see fluffy, white sheared DNA.

Modified from: "Generic, All Purpose DNA Extraction from Meat Protocol" Judy Brown

"Mammalian DNA Extraction" Theresa Knapp

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