Simultaneous measurements of mobility, dispersion, and orientation of DNA during steady-field gel electrophoresis coupling a fluorescence recovery after photobleaching apparatus with a fluorescence detected linear dichroism setup

B. Tinland, L. Meistermann, and G. Weill
Phys. Rev. E 61, 6993 – Published 1 June 2000
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Abstract

Orientation of molecules is responsible for the loss of separability during steady-field gel electrophoresis. In this work we develop a technique to measure simultaneously the relevant parameters involved in the separation mechanism: electrophoretic mobility, band broadening, and molecular orientation. To do that we have associated a fluorescence recovery after photobleaching (FRAP) apparatus with a fluorescence detected linear dichroism setup. This coupling allows one to follow the buildup of orientation during the FRAP experiment. Because orientation involves a change in the angular distribution of fluorescence, we have added a fluorescence polarization setup which can be used in parallel with the FRAP and gives an exact value of the steady-state orientation factor. We illustrate the possibilities of these combined experiments by analyzing the coupling of electrophoretic transport and orientation of λ DNA in 1% agarose gels.

  • Received 5 January 2000

DOI:https://doi.org/10.1103/PhysRevE.61.6993

©2000 American Physical Society

Authors & Affiliations

B. Tinland, L. Meistermann, and G. Weill

  • Institut Charles Sadron, CNRS, 6 rue Boussingault, 67083 Strasbourg, France

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Issue

Vol. 61, Iss. 6 — June 2000

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