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.

 


© 2002-2014 UW E. coli Genome Project