21 May 2011

Thymus DNA Extractions


Materials

    Fresh thymus
    Blender
    Beaker
    Sugar
    Pipet
    Centrifuge tube with cap
    Bufferin (325mg)
    Knife
    Graduated cylinders (10ml,100ml)
    Epsom salts
    Distilled water
    Centrifuge
    95% ice cold ethanol
    15 ml test tubetest tube rack or beaker
    Palmolive detergent
    Non-iodized salt

Solutions
Prep buffer solution:

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

10% detergent solution:

    90 ml distilled water
    10 ml Palmolive detergent

Salt solution:

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

Protocol

    Cut out a chunk of liver or thymus 1 inch square and place in the blender.

    Add 100 -150 ml prep buffer and 10 ml detergent solution to the blender.

    Blend for 1 minute or until the mixture is smooth.

    Pour the mixture into a beaker.

    Transfer 1 ml of the mixture to a centrifuge tube.

    Add 2 ml of salt solution, cap, and shake for 2 minutes.

    Centrifuge for 7 minutes in a balanced centrifuge.

    Carefully remove the tube from the centrifuge and note the two layers:
        Lower layer - pellet
        *upper layer - liquid (supernatant) and what has the DNA in it.

    Pipet or carefully pour the liquid into a clean test tube.

    Pour 5 ml ice cold ethanol carefully down the side of the tube to form a layer.

    Let the mixture sit undisturbed for a minute or two.

    The DNA will float in the alcohol. The DNA of the thymus will be long threads that easily spool.

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

"Mammalian DNA Extraction" Theresa Knapp 

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