Southern blot troubleshooting - Strange background issue (Jul/12/2005 )
I have been doing genomic DNA Southern blots using the alkaline transfer method and hybridization in a formamide-based buffer. However, when I develop the film, part of the blot appears to have a much darker background. The interesting thing is, it shows up across the whole blot (marker lanes, empty lanes, etc), but stops abruptly at a point from top-to-bottom. Hard to explain, but it results in a blot with a block of darker background at the top of the gel, but normal levels towards the bottom. Also, I have repeated, and the "line" where it stops differs between the two, so it doesn't seem to be a molecular weight issue of any sort. It seems to me to be a gel issue, I thought of ethidium, but I don't include EtBr in my gel or buffer while running it.
Anyone ever seen anything like this or have any suggestions??
Thanks so much,
Could you have a defect in your intensifyig screens? Could it be in the film processing?
I have seen this before,
When your blot is in the hyb bottle, does it overlap with itself? the border you are seeing could well be where the edge of your blot overlaps with itself.