Phusion green high fidelity dna polymerase

Genomic DNA was extracted from 10 million cells using the PureLink Genomic DNA Mini Kit according to the manufacturer’s instructions (Thermo Fisher Scientific). To exponentially amplify the HPRT1 gene, we designed primer pairs every 3–8 Kb (Additional file 2: Table S2). Amplicons were generated using 50 ng of gDNA in 50 μl reaction volumes containing 0.5 μM of primers. Loci 2 to 8 where amplified using the Phusion Green High-Fidelity DNA Polymerase (Thermo Scientific, Waltham, MA, USA) with the following parameters: 98 °C for 30 s, followed by 30 cycles of 98 °C for 10 s, 65 °C for 30 s and 72 °C for 3.5 min, and a final extension of 72 °C for 3.5 min. Locus 1 was amplified using the KAPA Long Range HotStart DNA Polymerase (KAPA Biosystems, Wilmington, MA, USA) with the following parameters: 94 °C for 3 min, followed by 40 cycles of 94 °C for 25 s, 60 °C for 15 s and 72 °C for 7 min, and a final extension of 72 °C for 7 min. PCR products were purified using the PureLink PCR purification kit according to the manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA, USA).

DNA amplifications for cloning and transformant screening were performed with Phusion Green High-Fidelity DNA Polymerase (Thermo) or Phire Green Hot Start II DNA Polymerase (Thermo), respectively. For cloning, the PCR products and digested plasmids were run on 1% or 0.8% agarose gels, and gel elution of DNA fragments was performed using Zymoclean Gel DNA Recovery Kit (Zymo). The in vitro assembly of PCR products was performed by using the In-Fusion HD cloning kit (Takara). The assembled plasmids were amplified by transformation of Stellar Competent Cells (Takara). Plasmids were isolated from overnight cultures grown at 37°C in LB medium containing 100 µg/mL Ampicillin using the NucleoSpin Plasmid isolation kit (Macherey-Nagel). The selection markers for fungal transformation were cloned into digested plasmids by using the Rapid DNA Ligation Kit (Thermo). The guide RNAs (gRNAs) were generated by EnGen sgRNA Synthesis Kit (NEB) according to the manufacturer’s protocol. The synthesized gRNA was purified by RNA Clean & Concentrator (Zymo) and assembled to the EnGen Spy Cas9 NLS (NEB) directly prior to the fungal transformation. Oligonucleotides utilized in this study are described in the Table S1.

To determine if G₁ larvae of Yellow-R2 and Hz-R2 that survived on diet surface treated with 1 μg Cry2Ab per cm² harbored mutations corresponding to sgRNA target sites, we amplified, cloned, and DNA sequenced the relevant HzABCA2 and HzTO gDNA corresponding to sgRNA target sites. We extracted gDNA separately from LAB-S, Yellow-R2, and Hz-R2 larvae using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). Phusion Green High-Fidelity DNA Polymerase (Thermo Fisher Scientific) was used with oligonucleotide primers (Supplementary Table S2) to amplify specific regions corresponding to both HzABCA2 and HzTO sgRNA target sites. PCR conditions were 98 °C for 1 min for 1 cycle; 98 °C for 5 s, 48 °C for 5 s, and 72 °C for 10 s for 35 cycles; 72 °C for 1 min; and hold at 16 °C. PCR amplicons were analyzed on 1.5% agarose gels and stained with SYBR Safe DNA Gel Stain (Thermo Fisher Scientific). DNA bands were excised, cloned into the pJET1.2 vector (Thermo Fisher Scientific), and transformed into One Shot TOP10 Chemically Competent E. coli (Thermo Fisher Scientific). Purified plasmid DNA was Sanger sequenced by Retrogen (San Diego, CA). Multiple sequence alignments were performed using MUSCLE^{43 (link)}. The full-length HzABCA2 sequence from LAB-S is deposited in the GenBank public database (Accession number OP186036.1).