EtBr in agarose gels vs. EtBr staining later

Richard M. Rohan rrohan at umabnet.ab.umd.edu
Thu Feb 23 11:47:28 EST 1995


On Thu, 23 Feb 1995 rmoldwin at midway.uchicago.edu wrote:

> 
> If I recall correctly, when I tried it, the EtBr migrated in the opposite 
> direction as the DNA.  Also, when you add EtBr to the gel, you can see that the 
> bottom of the gel is always darker than the top.  I think this is because the 
> EtBr migrates upward.  The implication is that low bp bands at the bottom of 
> the gel may not show up under UV light.  Am I right about this
> 
> 
This has also been my experience and the main reason that I do not favor 
incorporating EtBr into the gel solution.  Also EtBr alters mobility of 
supercoiled DNA, as already mentioned in this thread.  Given that 
staining times are quick and that EtBr solutions can be re-used, we 
prefer staining after the gel is run.  Also uses less EtBr and 
generates less waste.  Basic Methods in Molecular Biology (eds 
Davis, Dibner & Battey) recommends 0.1 mg EtBr per 100 ml gel.  We 
make a stock staining solution of 0.03 mg EtBr per 100 ml which lasts 
for several gels.  Also easier to dispose of 500 ml of stock solution 
every few months than contaminated running buffer after each gel.  
XC and BB dyes can be used to monitor run times on gels.  FMC, for 
example, publishes extensive charts correlating dye mobilities to DNA 
fragment sizes for all types of agarose gels.

Rich Rohan
University of Maryland




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