Phanta max super fidelity dna polymerase

Eight thousand sorted cells were harvested for genomic DNA extraction by addition of 20 μl of lysis buffer (Vazyme) following the manufacturer’s manual. For TIDER test, the genomic region in the vicinity of Cas nuclease target site was amplified by Phanta Max Super-Fidelity DNA Polymerase (Vazyme) using nested PCR. Purified PCR products were Sanger sequenced and analyzed as previously described^{44 (link)}. For deep sequencing analysis, the targeted genomic region was amplified by Phanta Max Super-Fidelity DNA Polymerase (Vazyme) using nested PCR, primers with barcode were used. PCR products were purified by Gel extraction kit (Vazyme) and sequenced on an Illumina HiSeq X System (150-bp paired-end reads). Forward reads were aligned to the reference sequences using BWA (v0.7.17-r1188) with parameter of “bwa mem -A2 -O3 -E1”. At each target, editing was calculated as the percentage of total reads containing desired edits without indels within a 10-bp window of the cut site. The target site informations are provided in Supplementary Table 2.

To isolate flanking sequences, specific nested primers were designed based on the upstream sequence of ltp promoter, downstream sequence of ZmMDH cDNA and downstream sequence of OCS terminator, separately. The primers used for experiments are listed in Table 2. In the first PCR reaction, the 25 μl reaction mixture included 1 μl of the cyclic digestion and ligation product, 12.5 μl 2× Phanta Max Buffer, 0.5 μl 10 mM dNTPs, 0.5 μl AP1 primer, 1 μl LSP1 or MSP1 or OSP1primer, and 0.5 μl Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech). The PCR program was as follows: 3 min at 95 °C, followed by 5 cycles with 15 s at 98 °C, 3 min at 72 °C, and 20 cycles of 15 s at 98 °C, 30 s at 56 °C, 3 min at 72 °C, and a final extension step of 5 min at 72 °C. In the second PCR, the 50-μl reaction mixture included 1 μl of a 20-fold dilution of the first PCR product, 25 μl 2× Phanta Max Buffer, 1 μl 10 mM dNTPs, 2.0 μl AP2 primer, 2 μl LSP2 or MSP2 or OSP2 primers, and 1 μl Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech). The PCR cycles were as follows: 3 min at 95 °C, followed by 35 cycles of 15 s at 98 °C, 30 s at 68 °C, 3 min at 72 °C, and a final extension step of 5 min at 72 °C.

The procedure for dPCR-CRISPR/Cas12a assay was similar to the dRAA-CRISPR/Cas12a assay. Briefly, dualplex PCR assay was carried out using Phanta® Max Super-Fidelity DNA Polymerase (P505-d1; Vazyme Biotechnology Co. LTD., China) in a 50 μl reaction mixture, containing 25 μl of 2 × Phanta Max Buffer, 1 μl of 10 μm forward and reverse primers (total of 4 μl; Supplementary Table S1), 1 μl of dNTP Mix (10 mm each), 1 U Phanta Max Super-Fidelity DNA Polymerase, 2 μl of genomic DNA, and 17 μl of H₂O. 2 μl of dPCR product was then added to the reaction mixture containing the ssDNA-FQ and Cas12a-crRNA complex.