Abstract:

We used magnetic tweezers to exert a constant force to separate double stranded lambda-phage DNA as a function of temperature and buffer content. The separation was performed at temperatures ranging from 20 to 50 degrees C in various Mg2+ buffers, including a T4 ligase buffer and a PCR buffer. At 30 degrees 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 degrees C.