What you see in the link above is an image for a 2% agarose gel in 0.1X TBE buffer run for 30 minutes at 23 V/cm. The separation distance is about 15 cm. The bands are for a ladder (300,280,260,240,220,200,180,160,140,120,100,80,60,40,20). The 20 bp moved out of the gel, but that's ok. The problem is about these two lanes on the right with the "diagonal" bands. The other lanes are ok and I think it's an achievement to separate such small and closely spaced bands using just 2% agarose within just 30 minutes. However the last few lanes on the right always annoy me with that appearence. Also it's not just about the shape of the bands. As you can see there is a massive decrease in mobility. It appears that these bands move slower than the others on the left lanes, especially the small bands (please compare the bright 100 bp bands). It happens over and over. I make fresh buffer everytime because I know I am using a highly diluted buffer that can get consumed quickly. The current is just 65-70 mA and I think the temperature of the gel stays below 40 C during the run. By the way I pour the gel in the lower right corner of the tray. I pour it very hot so that it does not solidify. Yet I still get these diagonal irregular and fuzzy bands. If it's about heat then why are the left edge lanes never affected? I have loaded equal volumes in the wells. The loaded volume is 1/4 the capacity of the well and there is about 30-60 ng per band. It's very strange. I always get this same problem with last few lanes on the right side (always the right). Just why doesn't it happen on the left side? is it because I pour the gel in the right side? but it's not a concentrated gel (just 2%) and I pour it while it's very hot. So I don't think there's something physically wrong with the matrix of the gel.
Any help would be appreciated.
Edited by Bassaml7, 16 May 2011 - 06:56 AM.