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
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
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.

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 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.