100414.220 at compuserve.com (Stefan Kley) wrote:
>Hi there,
>>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?
>
Have you tried incorporating the EtBr in the gel before polymerisation?
The visualisation will be much better.
The faint signal may be a result of the old EtBr since the affinity is the same,
the new solution will not remove the old from your DNA.
Good luck.
>
