Hello,
I am measuring the melting temperature of two hybridized oligomers (23 bp) with the steady state fluorescence in the spectrofluorimeter. In one strand I have a fluorophore (AF488) and in the other a quencher (BHQ), that is how it can be measured the Tm (when the temperature raises the signal too).
The thing is that when I put the DNA in presence of Ionic Liquids or DESs I observe always a melting transition at 30 ºC, 10 times less intense than the melting transition of the DNA which is also present around 50-60 ºC.
In different articles this melting transition also appears but they never explain why. Any idea?
I do not understand much about it, but you may be observing a transition from one solid state to another (a different molecular order) at that lower temperature.
The low temperature transition could be a melting from the ends event (nucleation or
so called premelting with longer DNA) followed by zipper opening and strand dissociation (main peak) .It could also be an order -order transition like @daniel
suggested.All these melting phenomena depend on the length and the precise sequence of the DNA as well as the type and concentrations of the ions present due
to differential stabilities of A.,T versus G.C pairs .What is the sequence and ionic environment in your experiments?
Could that just be a conformational change in the DNA induced by the temperature?. Perhaps a good control would be running the same experiment in aqueous buffer. Also, I'd run the melting by UV or CD, which would provide direct information about base pairing and conformation, whereas in the fluorescence experiment you're introducing extra variables (e.g., thermal quenching, conformational/orientation effects in the fluorophores, etc.)
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