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CTAB Procedure
作者:未知 来源:生物秀 时间:2007-7-24

    In the hood:
    96 well dish with bacteria
    titertech
    microtubes
    glass pipette

    Remove colonies from each well using the titertech and place them into the cover
    Pipette up and down to thoroughly mix the colonies
    Aliquot 300 µl of the culture into a microtube; save about 4 or 5 tubes

    In the lab:
    Centrifuge the culture for about 2 minutes or until a pellet has formed
    Remove the supernatant
    Resuspend the pellet in 576 µl TE, 15 µl of 20% SDS and 3 µl of 20 mg/ml proteinase K
    Incubate for 1 hour at 37 ℃
    Add 166 µl of 3M NaCl and mix thoroughly
    Add 80 µl of 10% CTAB in 0.7 M NaCl and thoroughly mix
    Incubate for 10 minutes at 65 ℃

    In the hood in the back:
    Add an approximately equal volume of chloroform (700 µl)

    In the lab:
    Centrifuge for 5 minutes at room temperature

    In the hood in the back:
    Remove white interface (should be able to be done by using pipetter)
    Transfer supernatant to another microtube
    Discard remaining solution in chloroform waste receptacle
    Add an equal amount of phenol/chloroform

    In the lab:
    Centrifuge for 5 minutes

    In the hood in the back:
    Transfer supernatant to a new microtube
    Discard remaining solution in proper waste receptacle

    In the lab:
    Add 0.6 vol of isopropanol and gently rock back and forth until white precipitant forms
    Centrifuge for 30 minutes
    Remove supernatant and wash pellet with 70% ethanol
    Centrifuge the tube for 5 minutes
    Remove supernatant
    Redissolve the pellet in 100 µl TE/10

     

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