Bands near edges running faster - (Dec/18/2013 )
I'm running a 10% native PAGE with ~100bp DNA at 150V in 1X TBE, and my bands (including dyes) are not migrating together. Yesterday (at 100V), just one side was running faster than the other. Today, both sides run faster than the middle. The bands toward the edges are also moving toward the center, i.e., are not going straight down. Any suggestions?
Usually if the bands are running faster on one side, the buffer tank is leaking or on an angle and one side hasn't got buffer completely covering it.
Sides running faster than the middle is probably due to overheating of the gel at the edges. It could also be because the gel is thinner at the edges than the middle, especially the case if you are using bulldog clips to hold the plates.
Bands not running straight could be a number of reasons, but the most common is probably too much salt in the sample.
bob1 on Thu Dec 19 01:10:59 2013 said:
Sides running faster than the middle is probably due to overheating of the gel at the edges.
Why are the edges of the gel overheating?
does your apparatus include cooling? you may be cooling the center more than the edges.
I don't believe so... I'm using a Biorad mini-protean tetrad system, which is why I don't think buffer coverage could be an issue. Actually what I'm starting to suspect is a combination of incomplete polymerization (I'm using fairly old reagents) with submerging the gel too soon, so that the buffer weakens the edges but not the middle. I'm new to all this, so still learning :)