Nytran supercharge

911 cells (1 × 10⁶) were transduced with either LV(GFP) or NILV‐S/MAR(GFP) at 1 IFU/cell, and single cell clones were generated through limited dilution. Genomic DNA was isolated from established clones using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). The DNA was digested with EcoRI (a restriction enzyme that cuts once in the LV or NILV‐S/MAR vectors) before being resolved on agarose gel and transferred to a positively charged nylon membrane (Nytran SuPerCharge, Whatman, GE Healthcare Life Sciences) by downside alkaline transfer using TurboBlotter (GE Healthcare Life Sciences) according to the manufacturer's instruction. The membrane hybridization and detection were carried out essentially followed the instruction of the digoxigenin (DIG) DNA Labeling and Detection kit (Roche, Basel, Switzerland). In brief, the detection probe, against the whole viral genome, was prepared from a plasmid by digesting the DNA and labeling it with DIG‐dUTP using Klenow fragment. Hybridization was carried out at 42°C for 16–18 h. The insoluble color development was generated using NBT/BCIP as substrate and the membrane was finally imaged using the GelDoc Imager (Bio‐Rad, Hercules, CA).

Collection of eraser crumbs (c. 15 mg; Mars^®plastic, Staedtler) was performed as described in [2 (link)] with the exception, that eraser crumbs were not collected on paper sheets but were transferred directly from the sampling material into 1.5 ml tubes.
DNA sampling via positively charged nylon membranes (Nytran^®SuPerCharge, Whatman, GE Healthcare) was performed once with dry membranes and once with membranes moistened with 0.5 M EDTA (pH 8.0). Membranes were cut into pieces (c. 1 x 2 cm), pressed for 30 s on the material and transferred to 2 ml tubes containing lysis buffer.
Eraser crumbs and nylon membranes were incubated for 3h at 55 °C in a lysis buffer containing 0.67 M EDTA (pH 8.0) and 1% SDS and were extracted using one volume of chloroform and isopropanol precipitation as outlined above.