Negative stain electron microscopy

RAD-51 and RFS-1/RIP-1 in indicated concentrations were incubated with 250 nM (in nucleotides) 150-mer poly(dT) ssDNA in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl₂, 2 mM ATP for 5 min °C. For negative staining, Quantifoil R2/2, 2 nm carbon, 400 Cu mesh grids were glow discharged for 30 s at 25 mA with a K100X glow discharger (EMS), 4 μL of sample was added to the grid left for 1 min. Excess sample was blotted away leaving a thin film. Then the grid was dipped into buffer solution twice and dipped twice into 2% uranyl acetate solution, blotting in between. Negative stain EM data were acquired on Tecnai Spirit TEM operated at 120 kV, equipped with a n FEI Eagle CCD camera.