Endo free plasmid maxi prep kit

The 27th immunoglobulin-like (I27) domain of muscle protein titin (schematically represented in Fig. 1D) was PCR amplified with sequence-specific primers containing PasI-modified ends and cloned so that one, two, and four copies of I27 (22 (link)) were obtained. The I27 domains were unidirectionally cloned into a unique PasI site (residue 29) of d37-41gp42 in the pCAGGS plasmid. The linkers were cloned just downstream of the transmembrane domain which ends at residue 22 and upstream of the gHgL binding domain which begins at residue 44. Other functional regions of gp42 were not disturbed. The ligated products were transformed into competent Escherichia coli DH5α and selected on plates containing ampicillin. DNA was isolated from overnight cultures using the Qiagen miniprep kit, digested to confirm the presence of the insert and correct orientation, and sequenced with primers internal and adjacent to the site of insertion and in both directions by the Northwestern Genomic Core Facility to confirm the resulting sequences. The resulting linker insertion sequences are shown in Table 1. Large-scale DNA preparations were isolated using Qiagen Endo-Free plasmid maxiprep kit and used in subsequent experiments. EBV gL in pCAGGS was previously described (28 (link)).

All plasmids were purified using the Qiagen Endo-free Plasmid Maxiprep kit. Plasmids were nucleofected into iPSC cells using the Lonza 4D-Nucleofector X unit (Buffer P3, Program CB-150) according to manufacturer's instructions. For each 20 μl nucleofection reaction, 0.2–0.5 × 10⁶ iPSC were transfected with up to 4 μg of plasmid DNA. Post-nucleofection, iPSC were plated onto 24- and 96-well Matrigel-coated plates containing mTesr1 media plus 10 μM Y-27632.
For CRISPR-based nucleofections with a targeting vector (Figures 1 and 2, and Supplementary Figures S2–S7), 2 μg of targeting vector plasmid, 0.5 μg of Cas9 plasmid, and 1.5 μg of total sgRNA plasmid were used. When two sgRNAs were used, 0.75 μg of each plasmid was combined. When no sgRNAs were used, 1.5 μg of pUC19 was used instead.
For CRISPR-based nucleofections without a targeting vector (Figure 3 and Supplementary Figures S10 and S11), 2 μg of total plasmid was used: 0.5 μg of Cas9 plasmid with 0.75 μg of each sgRNA plasmid or pUC19.
For TALEN-based nucleofections with a targeting vector (Supplementary Figure S5), 2 μg of targeting vector plasmid plus 2 μg total of TALEN plasmid was used. For one dsDNA break using one TALEN pair, 1 μg of each TALEN-expressing heterodimer plasmid was used. For two dsDNA breaks using two TALEN pairs, 0.5 μg of each TALEN-expressing heterodimer plasmid was used.