I have run a few electrophoretic mobility assay gels recently using Hoechst-33258 and ethidium bromide in order to observe the changes in the mobility of the DNA when each are used in comparison to a blank sample. In the case of EB, the mobility of the DNA is reduced due to the extension of the DNA helix on intercalation, which makes sense to me, but when I run H-33258, I see an enhancement of the mobility of the DNA, which is what I don't understand. Is this observation a common occurence?
I'm hopefully going to use these gels as diagnostic tools for studying the interactions of a novel compound with DNA so I'd just like to make sure I understand what is actually happening with my standards
Are you using linear or covalently closed circular DNA species? If the latter, then dye intercalation will produce positive supercoiling, which will change electrophoretic mobility.
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