We separate double stranded lambda phage DNA by applying a fixed force at a constant temperature ranging from 15°C to 50°C, and measure the minimum force required to separate the two strands, providing the first experimental determination of the phase boundary between single stranded DNA and double stranded DNA in the temperatureforce plane. The measurements also offer information on the free energy of dsDNA at temperatures where dsDNA does not thermally denature in the absence of force. While parts of the phase diagram can be explained using existing models and free energy parameters, others deviate significantly. Possible reasons for the deviations between theory and experiment are considered.

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We used magnetic tweezers to exert a constant force to separate double stranded λ-phage DNA as a function of temperature and buffer content. The separation was performed at temperatures ranging from 20 to 50 °C in various Mg2+ buffers, including a T4 ligase buffer and a PCR buffer. At 30 °C and pH 7.4 (10 mM Tris), we measured the unzipping force as a function of concentration for Mg2+ concentrations between 0.2 and 50 mM, and determined that the unzipping force is proportional to the logarithm of concentration. For comparison, we performed the analogous experiment as a function of Na+ concentration and found that the unzipping force is also proportional to the log of concentration, but requires a much higher cation concentration to achieve the same unzipping force as in Mg2+ buffer. We also constructed the phase diagram in the force–temperature plane for the unzipping in 10 and 50 mM MgCl2 at pH 7.4 (10 mM Tris). The phase diagram for 10 mM Mg2+ is similar to the one measured previously for phosphate buffer saline (PBS) but the phase diagram for 50 mM Mg2+ deviates significantly from those for 10 mM Mg2+ and PBS at temperatures between 20 and 35 °C.