Polycation-DNA complexes for x-ray scattering experiments was prepared using calf-thymus DNA (CT-DNA) as follows. Polycation solutions (1 mg/ml) was added to ~250 µg of CT-DNA (1 mg/mL) in 10 mM Tris-Cl (pH 7.5) for each x-ray experiment. A sufficient amount of polycation was added stepwise to ensure complete condensation. The resulting fibrous polyplex samples were thoroughly mixed by vortexing and spun down. Condensates were then transferred to 10 mM Tris-Cl containing ∼ 10 µM excess polycation solution. Samples were measured approximately 1 hr after preparation. X-ray scattering experiments were performed using a graded-multilayer focused Cu Kα radiation (1.54 Å) from a Nonius FR-591 rotating anode fine-focus X-ray generator operating at 45 kV and 20 mA. Collimation of the primary beam used a fine rear aperture beam tunnel. Samples were sealed with a bath of buffer solution (10 mM Tris-Cl) in the sample cell and then mounted into a custom sample holder at room temperature. Sample to detector flight path was filled with helium gas to minimize air scatter. Diffraction patterns were recorded with a PT-135 CCD detector, with phosphor optimized for Cu Kα radiation. Images were analyzed with Fit2d and OriginPro software. Calibration of the SAXS sample-to-detector distance was performed using silver behenate powder and found to be 23.2 cm. Bragg scattering peaks were used to determine interaxial DNA-DNA spacings. For a hexagonal lattice, the relationship between the Bragg spacing and the actual interaxial distance between helices (Dint) is calculated as Dint = (2/√3)DBr. For different samples equilibrated under the same conditions, Dint values were reproducible to within ∼ ±0.1 Å. No significant sample degradation was observed due to Xray exposure. Typical exposure times were 120 s.