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 H₂O 14.9

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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 H₂O 17ul Klenow Fragment 50u/ul 1ul

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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 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.