Cacodylic acid

Buffer equilibration for DNA with the appropriate buffer was carried out using Amicon Ultracel-30K filters (Millipore, MA), replacing Microcon YM-30 (Millipore, MA) used previously. Samples were prepared in 2 mM Na-EPPS, Rb-EPPS, or Cs-EPPS, pH 8.5; Rb-cacodylate was prepared by titrating RbOH (1 M, Aldrich, USA) with cacodylic acid (0.5 M in 2 mM Rb-EPPS buffer, Sigma, USA) to pH 8.5. The concentration of 24bp was varied from 0.2 to 2 mM. As the accuracy of the ion counting method at high salt concentrations depends on the dsDNA concentration, higher DNA concentrations were used at these salt concentrations, and experiments were carried out to demonstrate that final ion counting values were independent of the experimental DNA concentration (Figure S1).
DNA-containing samples (500 μL) were spun down to ∼100 μL at 7000g in Amicon Ultracel-30K filters (Figure 2, i). To minimize solution evaporation, experiments were conducted at 4 °C.^{60 (link)} Equilibration between ions associated with DNA and the bulk ions was shown to be complete after five rounds of the buffer exchange (Figures S2 and S3). dsDNA remained in the top chamber (Figure 2) throughout the equilibration buffer process; no DNA was detected in flow-through samples.