Genomic dna extraction kit

sgRNA and Cas9 protein were introduced by electroporation using NEPAGENE (NEPA21, Nepa Gene Co., Ltd. Chiba, Japan) as the ribonuleoprotein (RNP) complex. Briefly, the RNP complex was formed by mixing 30 μg Cas9 protein with 25 μg sgRNA and incubating the mixture at room temperature for 10 min. These were then electroporated to 1 × 10⁶ hiPSC with 100μL of Opti-MEM Medium (31985062; Thermo Fisher Scientific, Grand Island, NY, USA). At least 72 h after transfection, genomic DNA (gDNA) was extracted using the Genomic DNA Extraction Kit (MN740230.250; Macherey-Nagel, Allentown, PA, USA) according to the manufacturer’s protocol. To identify the gene editing efficiency and exact sequence edited, we utilized the IN/DEL analysis service for CRISPR validation (ATC-0120, Bioneer). In addition, correction of targeted genes was confirmed by AccIII restriction digestion of PCR products before and after gene correction.

DNA was extracted from 100 μL blood using the Genomic DNA Extraction kit (Macherey-Nagel, Düren, Germany) and eluted in an equivalent volume of elution buffer. DNA was screened for P. falciparum using ultrasensitive qPCR that amplifies a conserved region of the var gene acidic terminal sequence (varATS) according to a previously published protocol [41 (link)]. The varATS gene assay amplifies ~ 20 copies/genome [41 (link)]. The qPCR results were converted to varATS copies/μL using external standard curve of ten-fold serial dilutions (5-steps) of 3D7 P. falciparum parasites quantified by droplet digital PCR (ddPCR) [42 (link)]. The ddPCR thermocycling conditions, sequences and concentration of primers and probe are given in additional file 1. Asexual parasite densities were calculated by dividing varATS copy numbers by 20, reflecting the approximate number of copies per genome.
For all the gametocytes assays, RNA was extracted using the pathogen Nucleic Acid Extraction kit (Macherey-Nagel, Düren, Germany) and eluted in 50 μL elution buffer, i.e., RNA was concentrated two-fold during extraction. RNA samples were DNase treated (Macherey-Nagel, Düren, Germany) to remove genomic DNA that could result in a false positive pfs25 signal [43 (link)]. A subset of RNA samples was tested by varATS qPCR, and all tested negative.