Max super fidelity dna polymerase

The genomic DNA of fourth instar larvae of P. xylostella was extracted using the MEGA Bio-Tek Tissue DNA kit (Omega, Norcross, United States), followed by purification after RNase treatment. The sequence of the candidate Pxyellow gene was obtained from P. xylostella genomic database (http://iae.fafu.edu.cn/DBM/index.php). The specific primers for PCR amplification were designed by using the NCBI database’s primer tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The 2.3-kb fragment of the Pxyellow gene, including the gRNA target site, was PCR amplified with designed primer Yts-F and Yts-R (Supplementary Table S1). The PCR reaction was prepared by using the Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China) and carried out with the following conditions of 95 C for 3 min; 32 cycles of 95 C for 10 s, 62 C for 20 s, 72 C for 2 min; then 72 C for 5 min; and 4 C forever. The PCR amplified products were purified with the Omega Gel Purification Kit (Omega, Norcross, United States) following its protocol. The purified PCR product was sub-cloned into the PJET1.2 blunt-end vector (Thermo Scientific, Waltham, MA, United States) and confirmed through Sanger sequencing.

Briefly, 48 h post-injection, approximately 100 embryos were collected into a 1.5 mL tube and frozen immediately with liquid nitrogen. Honeybee embryo DNA was isolated and purified using the phenol–chloroform extraction and alcohol precipitation method [20 (link)]. The amplicon of the intended targeting region was amplified from approximately 70 ng genome DNA template using Max Super-Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China) with the site-specific primers (Table S1) for Amyellow-y. Thereafter, the purification of PCR products was performed with the Omega gel extraction kit (Omega, Guangzhou, China). Cloning was carried out using the pEASY-blunt vector (TransGene Biotech, Beijing, China) to analyse the mutagenesis efficiency and types. Then, 100 individual clones were picked randomly and subsequently sent to Biosune Biotechnology Company (Fuzhou, China) for Sanger sequencing. For several target sites where any mutation was not detected via Sanger sequencing, the deep sequencing of site-included PCR amplicons was conducted on an Illumina MiSeq platform (Sangon Biotech, Shanghai, China). The number of reads for the deep sequencing was ~40,000. Sequence alignments were carried out with BioEdit8.1.0 software.

Quantitative PCR (qPCR) was performed to quantify the expression of uracil phosphoribosyltransferase [(UPRT) (TGGT1_312480)] gene and apicoplast gene (TGGT1_302050) between WT strain and ΔFabZ strain. The 18 s rRNA gene was used as an internal reference for normalization. The qPCR assay was performed as described previously (Wu et al., 2009 (link); Yeh and DeRisi, 2011 (link); Reiff et al., 2012 (link); Bansal et al., 2017 (link)). Genomic DNA was isolated from freshly egressed tachyzoites using Tiangen DNA extraction kit according to the manufacturer’s directions. The apicoplast and UPRT-specific gene sequences were amplified using Max Super-Fidelity DNA Polymerase (Vazyme). The amplification reaction mixture included 10 μl of 2 × SYBR Green pro Taq HS Premix (final concentration 1 ×) (Accurate Biology), 0.4 μl of 10 μM of each primer, 40 ng of the extracted T. gondii DNA, 0.4 μl of ROX Reference Dye, and sterile water to a final volume of 20 μl. qPCR was performed using an initial denaturation at 95°C for 30 s; followed by 40 cycles of amplification at 95°C for 10 s, 56°C for 20 s, and 72°C for 30 s.