danielg@uncmed.med.unc.edu (09/21/90)
Recently I posted a note concerning various problems with Southern blot autoradiography, and had a few responses. My main problem is "Denhardt's spots". The following suggestions have come in, and I will include my comments. Am hoping for more suggestions, explanations, and encouragement -there *has* to be a way to eliminate these @#!*&%@! spots. 1. Agarose stuck to the filter after transfer: It was suggested that I manually, lightly rub the surface of the filter after transfer, before baking, to remove agarose particles that would cause spots on the autorad. I objected to this at first b/c the spots were so round (looked like bubbles, not blobs of squished agarose), but the writer suggested that the pieces were so small that I couldn't see their true shape. However, I discarded this theory b/c after we stripped the filter and reprobed, the spots showed up in diffent places on the filter. 2. Bubbles in pre-hyb/hyb: This was a good suggestion, or at least, it lined up with our thinking. However, we carefully pre-hybed and hybed, squishing the solution around in the bag every so often, to move any bubbles that might be stationary. The spots did not even diminish. Blah. 3. Precipitation of Denhardt's onto the filter: Some have suggested that when you transfer the filter from hyb to wash solution, that the Dextran sulfate, or some other component (nonfat dried milk, if your solution contains that, but don't quote me, I'm so tired of figuring out what does what in the hyb solution, I need a vacation!) precipitates onto the filter (due to temperature change? Doesn't formamide freeze near RT?) and must be manually removed by rubbing membrane after first wash. We are trying this now. 4. Poor washing: We tried washing the filter longer, but band intensity decreases as much as the noise, so the probe is stuck just as well. One writer suggested that when you dilute the Dehardt's by placing the filter in the first wash, the probe is able to stick to the blot where ever it likes. He suggested that the first wash be vigourous. Any comments. I suppose I could do a little more thinking before I post, so if I have overlooked something obvious, please have mercy! All help welcome, rescue us from this terrible malady, I see spot before my eyes! dan ___________________________________________________________________________ **** The shallow man has *** | Do not be overly righteous, nor overly **** has opinions; *** | wise: why should you destroy yourself ? **** the deep, *** | - King Solomon, wisest man of his **** convictions. *** | day, Ecclesiastes 7:16.