I have a problem with ethidium bromide. I ran a PAGE today to check
the integrity of a few cDNA probes that I'm about to use in Northern
Hybridisations. In order to visualize the DNA with UV light, I put the
gel into an ethidium bromide bath for 45 minutes after the run.
However, under UV light, the gel appeared empty, lacking a signal even
from the slot where I loaded the DNA standard.
Seeing the EtBr solution I was using was quite old, I prepared a new
one and put the gel through another 45 minutes of bathing in this new
solution (diluted to 0.5 mg/l). Back on the UV screen, the DNA
standard's bands were now clearly visible, my cDNAs (60 ng loaded)
gave only very faint signals, however.
Now, my question is: does this result really tell me that my cDNAs are
degraded (which I consider a clear possibility, given their age). Or,
could the fact that their signals were faint result from the "old"
ethidium bromide, or fragments thereof, having intercalated into the
DNA and, although not being visible in UV light, blocking the "new"
EtBr from binding?
100414.220 at compuserve.com
Benjamin Franklin University Hospital
Berlin - Germany