DNA "refusing" to be run in agarose gel

Peter Kaplan kaplan at research.nj.nec.com
Wed Aug 30 16:22:01 EST 1995


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
experiment:

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
Result
     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
loading buffer.

ul = microliter
Promega blue/orange loading dye:
10% Ficoll
0.25% Bromo Blue
0.25% Xylene Cyanole
0.4%  Orange G
10mM  Tris/Cl pH 7.5
50mM  EDTA  pH 8.0



More information about the Methods mailing list