High fidelity dna taq polymerase

Genomic DNAs were isolated from the cotyledon explants (30 explants per sample) or mature leaves (three distinct leaflets from three different young leaves per plant per sample) using the CTAB method. The MiSeq sequencing service (MiniSeq^TM System, Illumina, San Diego, CA, United States) was used. MiSeq samples were prepared in three PCRs according to the manufacturer’s guidelines with genomic DNAs as templates for the first PCR. The first and second PCRs used primers listed in Supplementary Table 5, whereas the third PCRs were performed with the manufacturer’s primers to assign sample IDs. The first PCR primers were designed for binding to the upstream and downstream sequences from the homologous donor sequence junctions to avoid amplifying the donor DNA sequences. The second PCR primers were designed to amplify 150–180-bp flanking the targeted base changes at the targeted sites. High-fidelity DNA Taq polymerase (Phusion, NEB, Ipswich, England) was used for PCR. The MiSeq raw data FASTQ files were analyzed by the Cas-Analyzer tool (Park et al., 2016 (link)). The indel analysis window was set to 5 bases, with a comparison range covering both read ends. The GT efficiency was assessed using the corresponding donor sequence as the input HDR donor sequence.