Favorprep blood cultured cell genomic dna extraction mini kit

HEK293 cells (American Type Culture Collection [ATCC], Manassas, VA; cat. no. CRL-1573) and Hep G2 cells (ATCC; cat. no. HB-8065) were maintained in Dulbecco’s modified Eagle’s medium and Roswell Park Memorial Institute, respectively, supplemented with glucose (4.5 g/L), 4 mM glutamine, 1 mM sodium pyruvate, 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 mg/mL). Hep G2 cells were transfected with plasmids using Neon Transfection (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Briefly, 1.5 × 10⁵ cells were resuspended to 10 μL of R buffer in Neon Transfection kit. Subsequently, 1230 V/20 ms/3 pulses Neon system is used to electroporate the cells. Two days after transfection, the cells were harvested, and genomic DNA was extracted using a FavorPrep blood/cultured cell genomic DNA extraction mini kit (FAVORGEN). For transfection of HEK293 cells, 5 × 10⁴ cells were seeded the day before transfection in 24-well plates. The next day, Total 1 μg plasmids encoding human optimized F9 donor and CjCas9 was diluted to 200 μL Opti-MEM and mixed with diluted lipofectamine 2000 (1:2 ratio) and then incubated for 20 min and added to cells. Genomic DNA was isolated after 4 days of transfection.

The genomic DNA samples were prepared using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and the FavorPrep Blood/Cultured Cell Genomic DNA Extraction Mini Kit (FAVORGEN, Ping-Tung, Taiwan) to isolate genomic DNA from cells according to the manufacturer’s protocol. The genomic DNAs were dissolved in distilled water after purification to produce an isolated environment and to remove the noise from other residue. To determine the quantity of DNA obtained, we used a NanoDrop ND-1000 UV-Vis spectrophotometer (Wilmington, DE, USA). The concentration of the blood cancer DNA samples was 500 ± 4 µg/ml, and the concentration of 293 T, M-293T, and exposed M-293T DNA samples was 300 ± 7 µg/ml.