M7 PEG precipitation to concentrate pools of 6HB slats:

The slats as folded on 96-well PCR plates were collected and combined into pools using a manual multichannel pipette. Each pool maximally had ~100 slats, though the number of slats in a given pool was variable depending on the design of the megastructure. We generally kept the slats in one layer of the megastructure in a separate pool from the perpendicular slats in the other layer of the megastructure, so that slats with complementary handles would not be concentrated in the same mixture together. Our rationale was that spontaneous interactions between the slats during concentration could be deleterious to the yield, though we did not study this carefully to determine whether such care was necessary. The pooled slats were subsequently concentrated using two rounds of PEG precipitation, as adapted from as published previously⁴⁸. The Mg²⁺ in the slat pool was increased from 6 mM to 20 mM by adding the appropriate volume of 1 M MgCl₂. One volume of 2x PEG-purification buffer (5 mM Tris, 1 mM EDTA, 15% w/v PEG-8000, 510 mM NaCl) was added and mixed with the equal volume of pooled slats in a 2 mL round-bottom tube. The mixture was spun at 16k × g for 25 min, the supernatant was gently extracted using a pipette, and the pellet was resuspended in 50 μL 1x TE buffer with 20 mM MgCl₂. The second round of PEG precipitation was performed using an equal volume of 2x PEG-purification buffer and the final slat pellet resuspended in a small volume of 1x TE buffer with 10 mM MgCl₂ so that the total concentration of slats was ~2 μM. However, the volume was adjusted as needed so that the pool was sufficiently concentrated to achieve the desired final per slat concentration in Method 9 or Method 10. The final slat pool was placed on a shaking incubator set to 1000 rpm at 33°C for about one hour. Finally, the concentration of DNA was measured on a Nanodrop 2000c Spectrophotometer (Thermo Scientific^™) so as to estimate the final nM concentration of slats.