Top of agarose gel blank? - (Jun/24/2014 )
I'm running lots of PCR reactions for genotyping on a long gel (taller than it is wide) such that each gel accomodates 96 samples with 24 samples per row. Occassionally, but not always, I will find that the top row of the gel is blank after I run it while the rest of the gel looks beautiful. I can overexpose the gel and just see the marker and my positive control band, but the data are not interpretable and I have to rerun the samples. Do any of you have insight into how I can address this issue?
How are you staining the gel? Could the gel stain be migrating out of your gel? I've seen this happen in gels with ethidium bromide, but the migration happens from the bottom of the gel, not the top. Perhaps another dye runs in the opposite direction?
Depending on how long you run a gel, the EtBr can migrate out. I've had this happen when running a gel on higher voltage multiple times (I'd save the gel to run more samples in the unused wells later that day). I've had to stain the gel after the run to see anything even though I normally put the EtBr in the melted agarose when pouring the gel. Perhaps this is the problem?
I am staining with EtBr (added to the gel before it's poured at 10ug per 100ml of agarose). I know that it can migrate out of the gel, but as phage434 mentioned, this should happen from the bottom of the gel, not the top. I've had that happen as well, but this top-of-the-gel thing just doesn't make sense to me. If it happens again today, I'll put some extra EtBr in the running buffer and see if that fixes the issue.