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        CTAB Procedure

        互联网

        965
         
         

        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 %26micro;l 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 %26micro;l TE, 15 %26micro;l of 20% SDS and 3 %26micro;l of 20 mg/ml proteinase K
        Incubate for 1 hour at 37 ℃
        Add 166 %26micro;l of 3M NaCl and mix thoroughly
        Add 80 %26micro;l 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 %26micro;l)

        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 %26micro;l TE/10

         

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