Donut-shaped spot - Dot-Blot hybridization (Jul/26/2007 )
Hi everybody !!
I'm just starting Dot-Blot experiments. I'm using a minifold to spot the RNA on the membrane. RNA are previously denatured with Glutaraldehyde (in Phosphate buffer 50 mM pH7) and then diluted in DEPC water-Poly A and Bromophenol blue.
I tried to do a standrad curve with commercial E.coli RNA and with a universal probe. The spot after radioactivity detection have a donut shape. (see the attached picture) Maybe it's a problem of ununiform drying when I spot the RNA...
If anyone have an idea of the problem and how to improve the homogeneity of the spotts...
Thank you in advance
based on your pic, I can offer a few comments -
I think perhaps your assembly isn't tight enough; the sandwich is slightly loose and that's why you're not seeing discrete dots. perhaps your gasket is dirty, or wearing out? if not, maybe try to tighten it more?
as for the donuts, my best guess is that there is a small airbubble that is sitting over the hole in the well - so the fluid is being sucked down around the air bubble and all your goodies are sticking to the membrane around the rim of the hole.
tighten the seal, watch for airbubbles - maybe slow your suction rate just a bit? - and hopefully you'll get nice discrete little spots
wat aimikins says is right i guess also u can try to load it with buffer initially and then followed by ur samples. alternatively u can also try n increase the volume of ur samples loaded this ensures the nucleic acids come in contact with the membrane slowly and in uniform fashion.
hope this is ok....!