This thread began with a sample that wouldn't settle into its well;
rather it floated out of the gel and spread over the surface.
As an amateur working with microbiology, I was fascinated to read the
thread. A week before it began, I had the same problem. It turned out
that my sample had a pH of 4.6 and I was loading into a pH 8.4 TBE
buffer. I guessed that the sample pH should be close to the buffer pH
to avoid electrostatic forces from the boundary between pH 4.6 and
pH 8.4. Then I read the thread, where many people wrote:
>The fact that your DNA sample spreads out of the well can have the
>following cause in my opinion:
>>your sample contains traces of ethanol, parafin, or other traces of
>substances that don't belong in your DNA sample.
This answer bugs me, because I can't figure out why trace ethanol should
make an otherwise dense solution float. So, a few minutes ago I did an
Lane 1 2 3 4
W T W T 10 ul of W=dH2O or T=10mM Tris pH 8.4
- - E E - = notheing E = 2 ul ethanol
D D D D 2 ul 6x Promega Blue/Orange loading dye
fl s fl s fl=floats, s=sinks
My conlcusion is--- pH imbalance makes gel loading nearly imposible.
Ethanol contamination can make the situation worse
but this is a secondary.
This experiment does not rule out salt imbalance as a cause for floating
ul = microliter
Promega blue/orange loading dye:
0.25% Bromo Blue
0.25% Xylene Cyanole
0.4% Orange G
10mM Tris/Cl pH 7.5
50mM EDTA pH 8.0