DNA "refusing" to be run in agarose gel

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Wed Sep 6 10:47:05 EST 1995


 In article <422kpp$kts at shakti.nj.nec.com> 
 kaplan at research.nj.nec.com (Peter Kaplan) writes:

:  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

You are not using enough ficoll to change the density of the solution.
I always use 1/3 to 1/1 volumes of 1x dye solution = 10% ficoll/0.05%
Amaranth (red) in 1xTBE and I've never had it float. Try adding various
amounts of this solution (1-10 ul) to your 10 ul sample or water before
loading and see what happens.  My bet is that in the experiment above,
the salt in the sample changed the density and the pH has nothing to do
with it - except that if you've prepared the 10 mM Tris by mixing Tris-HCl
with water and then pH-ing it up by adding NaOH. This increases the NaCl
concentration and therefore changes the density!

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
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