Elsevier

Methods in Enzymology

Volume 180, 1989, Pages 304-325
Methods in Enzymology

[22] absorbance melting curves of RNA

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Publisher Summary

This chapter discusses the experimental methods needed to acquire a melting curve and the analysis and interpretation of the data. Any standard commercial UV spectrophotometer can be equipped to measure melting curves. A useful instrument is a single-beam Gilford (Oberlin, OH) spectrophotometer (Model 2530) with an automated reference compensator that allows melting curves to be obtained on three separate samples simultaneously. One major advantage of using UV spectroscopy is the high sensitivity of the method. Normally, the absorbance of the sample used should be between 0.2 and 2.0. Sample preparation for UV melting studies is straightforward. The RNA stock solution is prepared by dialysis against the desired buffer and different concentrations are made by dilution. The high salt concentration is chosen to minimize electrostatic repulsion between strands and to avoid divalent ions, which catalyze hydrolysis of RNA and favor triple-strand formation. This solvent provides a standard condition for measuring melting curves and for comparing results with previously published data.

References (31)

  • G. Felsenfeld et al.

    J. Mol. Biol.

    (1965)
  • K.J. Breslauer et al.

    J. Mol. Biol.

    (1975)
  • J. Gralla et al.

    J. Mol. Biol.

    (1973)
  • D.M. Crothers et al.

    J. Mol. Biol.

    (1974)
  • S.M. Freier et al.
  • I. Tinoco et al.

    Nature (London) New Biol.

    (1973)
  • A. Stein et al.

    Biochemistry

    (1976)
  • Calbiochem

    Buffers

    (1985)
  • M.T. Record

    Biopolymers

    (1967)
  • C. Stevens et al.

    Biopolymers

    (1964)
  • N.R. Kallenbach et al.

    Nature (London)

    (1983)
  • D. Poland et al.

    Theory of Helix-Coil Transitions in Biopolymers

    (1970)
  • I. Tinoco

    J. Am. Chem. Soc.

    (1960)
  • A. Rich et al.

    J. Am. Chem. Soc.

    (1960)
  • J. Applequist et al.

    J. Am. Chem. Soc.

    (1965)
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