Bge cells were harvested using a cell scraper, washed twice in Bge medium, counted, and resuspended at 20,000 cell/µl in Opti-MEM medium (Sigma-Aldrich, St. Louis, MO). Two (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted September 18, 2020. ; https://doi.org/10.1101/2020.04.07.029629 doi: bioRxiv preprint 9 million cells were transferred into 0.2 mm path length electroporation cuvettes (BTX Harvard Apparatus, Hollister, MA) containing 6 µg pCas-BgAIFx4 in ~100 µl Opti-MEM. The cells were subjected to electroporation using one pulse at 125 volts for 20 milliseconds, using a square wave pulse generator (ECM 830, BTX Harvard Apparatus). Immediately thereafter, the Bge cells were maintained in 12-well plates (Greiner Bio-One) at 26ºC. The mock control included Opti-MEM only for electroporation. The presence of transcripts encoding the B. glabrata actin and the Cas9 was monitored daily for nine days following transfection by electroporation (Fig. 1c).
The copyright holder for this preprint this version posted September 18, 2020. ; https://doi.org/10.1101/2020.04.07.029629 doi: bioRxiv preprint 9 million cells were transferred into 0.2 mm path length electroporation cuvettes (BTX Harvard Apparatus, Hollister, MA) containing 6 µg pCas-BgAIFx4 in ~100 µl Opti-MEM. The cells were subjected to electroporation using one pulse at 125 volts for 20 milliseconds, using a square wave pulse generator (ECM 830, BTX Harvard Apparatus). Immediately thereafter, the Bge cells were maintained in 12-well plates (Greiner Bio-One) at 26ºC. The mock control included Opti-MEM only for electroporation. The presence of transcripts encoding the B. glabrata actin and the Cas9 was monitored daily for nine days following transfection by electroporation (Fig. 1c).