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        Genomic DNA Labeling Protocol

        互联网

        1075

        We typically use 0.5ug of E. coli genomic DNA in a labeling reaction for each hybridization. The genomic DNA was fragmented to 500 to 1000bps before labeling. The following protocol should produce enough labeled probe for 8 hybridizations.

        For labeling 4μg Genomic DNA :

        DNA Mix

        Genomic DNA
        1.9μg/μl
        2.1μl
        Random Hexamer
        5mg/ml
        1μl
        H2 O
        14.9

        Total
        20μl

        Heat to 95℃ for 5min, place on ice for 5min

        Labeling

        DNA Mix
        20μl
        dAGC
        5mM each
        5μl
        EcoPol Buffer
        10x
        5μl
        CyDye-duTP
        1mM
        2μl
        H2 O
        17μl
        Klenow Fragment
        50μ/μl
        1μl

        Total
        20μl

        Incubate at 37℃ for 3.5 hours

        Add 2.5μl 0.5M EDTA to stop reaction

        Clean up Labeled Probes

        Prewash Microcon-30 microfilter by adding 450ml miliQ H2 O and spinning for 10 min. @ 12,000 RPM.

        Add 450ml miliQ H2 O to each of the probe samples (or total 500μl).  Mix thoroughly by pipetting up and down.  Transfer samples to separate Microcon-30 microfilters. (Amicon)

        Spin at 12,000 RPM in microfuge for 10 minutes or until 20-40 ml remains in the filter.

        Add 450 ml miliQ H2 O to the probe and gently mix by pipetting up and down.  Be careful not to touch the filter at the bottom of the filtration unit.

        Spin 10 minutes at 12,000 RPM.

        Repeat step 4 , spin 12min to get smaller volume.

        Invert column into a fresh tube and spin 1 minute at maximum speed to recover probe.  Carefully measure recovered probe volume if necessary.

        Probe can be stored at 4℃ or -20℃ in dark for further use.

        Reagents and Suppliers

        Cy3-dμTP 1mM Perkinelmer NEL578
        Cy5-dμTP 1mM Perkinelmer NEL579
        Klenow Fragment 50μ/μl NEB M0210M
        100 mM dNTP seundefined 10X Amersham 27-2035-01
        pd(N)6 Sodiμm Salt (Hexamer) 50μ Amersham 27-2166-01
        Microcon YM-30 colμmn Amicon 42410

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