This is a cached page for the URL (http://www.genome.wisc.edu/resources/protocols/genomiclabeling.htm). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Genomic DNA Labeling Protocol

 

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 UW E. coli Genome Project