For ECD assay sensitivity, 10-fold serial dilutions of NA samples (10-1 to 10-8) were made in buffer containing 40.0 µg yeast tRNA (Invitrogen) and 9.6 µg BSA (NEB) per 1.0 ml of dH2O.
Serial dilutions of proteins (in triplicates) were made in buffer F (20 mM HEPES pH 6.8, 150 mM KCl, 1 mM EDTA), and 20 nM (wild-type primase and ΔCTD-primase) or 80 nM (PriL-CTD) of fluorescein-labeled DNA oligos.
Four serial dilutions of the proteins of ¼ were made in buffer A (25 mM HEPES, 50 mM NaCl).
Measurements were made in buffer consisting of 20 mM Hepes/KOH (pH 7.2), 100 mM KCl and 1 mM CaCl2 or 5 mM EDTA.
All samples were made in buffer A. Vesicles for CD spectroscopy were extruded through 50-nm pore diameter filters to minimize the effects of light scattering, and spectra were measured in 0.1-mm path length cuvettes to further minimize scattering contributions.
For the protease assay, a 0.3%% azocasein (Sigma-Aldrich) solution was made in buffer B (50 mM Tris HCl, 0.5 mM CaCl2, pH 7.5). 1 mL of filtrate was added to 1 mL of azocasein solution which was then incubated at 37 °C for 15 min.
Gradients (10-ml total volume, 30-50%, w:v) were generated from a 50% sucrose solution made in D2O buffered to pH 7.2 with NaOH, and a 30% sucrose solution made in 10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 5.7 mM Na2EDTA.
At the end of the incubation, 10 μL of 5 mg mL 1 MTT solution (Sigma) made in phosphate buffered saline (PBS) (Gibco) was added to each well and incubated for minimum of 2 to 3 h until crystals formation was observed.
As described in Materials and Methods, changes were made in the buffer used for the passive diffusion of nuclei, in the salt concentration of the wash buffers, and in the conditions of the concentration step and the sucrose gradient.
