Southern Blot Insanity - (Oct/28/2008 )
I am attempting to verify the existence of a genetic mutation in a colony of chimeric mice I have presently. Because they are chimeric, I expected to see mostly WT with maybe a few HETs. I ran 29 samples in two different rows of wells on the same 1% agarose gel. The gel slices went through the same process together, denaturation, neutralization. They were transferred onto the SAME membrane in a blotting stack overnight- which did not dry out or anything. The membrane was cross-linked, hybridized with the same DNA probe, washed and so on all the same. When I developed my blot, the DNA that was on the bottom slice of the gel looks just as it should. I can see the bands for my WT and my HETs. The DNA that was on the top gel slice however looks ridiculous. It literally looks like the Polaroid image of the gel just after its been run on electrophoresis. The area where the gel was placed on the membrane is very dark, I can see the ladder perfectly as a set of light bands. The DNA appears in bright rows against the dark background. I cannot understand what I have done. Something would have had to happen before before the transfer to the membrane. Any suggestions would be greatly appreciated. I would have uploaded an image but the website wouldn't let me.
Did you use chemical cross-linking or a stratalinker? (i.e. could it be better crosslinked on one side than the other)...
When you hyb'd did you do it on a roller? (could it have hyb'd better on one side then the other - giving non-specific bands?).
It does sound weird though.
p.s. Southerns are hell!
It sounds like the membrane started to dry out on you.
I've had this sort of happen to me before. Are you using radioactive detection or non-radioactive detection?
If radioactive, sometimes the static electricity between the film and the membrane/enhancer screen affects how the film will develop afterwards. If the film was removed too quickly, especially after removing from -70oC, I got a black image on part of the autoradiogram.
If nonradioactive, then the membrane might have dried out while adding the chemiluminescent substrate. If the membrane dries out before adding the substrate, the substrate will bind to the whole membrane and produce dark background on the entire area that dried out. The same thing could happen if you add the substrate dropwise onto the membrane. The same thing will happen if the membrane dries out in the detection cassette (especially for exposure times in excess of 5-10min). You could try placing 20 drops of your substrate to a piece of acetate, making a little puddle onto which you lay your membrane (DNA side down) instead of applying substrate dropwise. Then cover the lower sheet of acetate with a top sheet of acetate while the membrane incubates with the substrate. You could also try adding a drop or two of detection buffer to the membrane as you assemble the detection cassette; this will add more moisture to the detection sandwich and hopefully prevent your membrane from drying out. Make sure you wipe out any bubbles and sort of push the detection buffer to the perimeter of the membrane so you form a wet seal surrounding the membrane.
That's about all I can think of right now.
I'm not sure how you obtained the negative image of the ladder :S
hope this helps