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:
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