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 set* 10X Amersham 27-2035-01 pd(N)6 Sodiμm Salt (Hexamer) 50μ Amersham 27-2166-01 Microcon YM-30 colμmn Amicon 42410
*for 10X stock: 5 mM each of dA, dG, dC.
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