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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 Heat to 95C for 5min, place on ice for 5min Labeling Add 2.5ul 0.5M EDTA to stop reaction Probe can be stored at 4°C or -20°C in dark for further use. Reagents and SuppliersGenomic DNA 1.9ug/ul2.1ulRandom Hexamer 5mg/ml1ulH2O 14.9Total 20ulDNA Mix 20uldAGC 5mM each5ulEcoPol Buffer 10x5ulCyDye-dUTP 1mM2ulH2O 17ulKlenow Fragment 50u/ul1ulTotal 20ulIncubate at 37°C for 3.5 hoursClean up Labeled ProbesPrewash Microcon-30 microfilter by adding 450ml miliQ H2O and spinning for 10 min. @ 12,000 RPM.Add 450ml miliQ H2O 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 H2O 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.Cy3-dUTP 1mM Perkinelmer NEL578 Cy5-dUTP 1mM Perkinelmer NEL579 Klenow Fragment 50U/ul NEB M0210M 100 mM dNTP set* 10X Amersham 27-2035-01 pd(N)6 Sodium Salt (Hexamer) 50U Amersham 27-2166-01 Microcon YM-30 column Amicon 42410 *for 10X stock: 5 mM each of dA, dG, dC.