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        Genomic DNA Labeling Protocol

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        Genomic DNA Labeling Protocol

        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 4ug Genomic DNA:

        DNA Mix

        Genomic DNA
        1.9ug/ul
        2.1ul
        Random Hexamer
        5mg/ml
        1ul
        H2 O  
        14.9

        Total  
        20ul

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

        Labeling

        DNA Mix  
        20ul
        dAGC
        5mM each
        5ul
        EcoPol Buffer
        10x
        5ul
        CyDye-dUTP
        1mM
        2ul
        H2 O  
        17ul
        Klenow Fragment
        50u/ul
        1ul

        Total  
        20ul
        Incubate at 37°C for 3.5 hours

        Add 2.5ul 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 500ul).  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°C or -20°C in dark for further use.

        Reagents and Suppliers

        Cy3-dUTP 1mM Perkinelmer NEL578
        Cy5-dUTP 1mM Perkinelmer NEL579
        Klenow Fragment 50U/ul NEB M0210M
        100 mM dNTP seundefined 10X Amersham 27-2035-01
        pd(N)6 Sodium Salt (Hexamer) 50U Amersham 27-2166-01
        Microcon YM-30 column   Amicon 42410

         

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