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