Transtaq hifi dna polymerase

Ptermoalus puparum embryos (<10 hr after parasitism), larvae (combined 2–3 day larvae after parasitism), yellow pupae (mixed with female and male pupae), adult females and males (combined 1–5 day adults after emergence) were collected and rinsed with PBS. Total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol and then reverse transcribed using TransScript One-step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China) with random primers. Isoform-specific primers were designed to span constitutive exon 7 and alternative exon 8 using PerlPrimer v1.1.21 [79 (link)] and are listed in S1 Table. PCR was performed using TransTaq HiFi DNA Polymerase (TransGen, Beijing, China) with 20–35 cycles of amplification. PCR products were analyzed by electrophoresis on a 1% (g/mL) agarose gel and confirmed by Sanger sequencing (Sangon Biotech, Shanghai, China).

To quickly identify the colonies containing the transformed aphVIII insert, Chlamydomonas colony PCR method was employed [30 (link)]. Briefly, cells suspended in 50 μl 5% (w/v) Chelex-100 (Bio-Rad, U.S.A.) were boiled for 10 min followed by immediately vortexed rigorously for 20 s. After on ice for 2 min, 1 μl of supernatant was prepared by centrifugation at 14000 rpm, 5 min for PCR template. The 264 bp DNA fragment was amplified by TransTaq HiFi DNA Polymerase (TransGen Biotech, China) with a forward primer F1 (5′-GATTCCCGTACCTCGTGTTG-3′) and a reverse primer R1 (5′-TCGTCCAGATCCTCCAAGTC-3′), using 29 cycles of thermal denaturation for 30 s at 97°C, annealing for 30 s at 58°C, and extension for 30 s at 72°C. PCR products were visualized and analyzed by running 1% (w/v) agarose gel and Gel Imaging Systems (Bio-Rad, U.S.A.).

For recombinant expression of PpSerpin-1 isoforms, constitutive fragments without signal peptides and alternative fragments were separately amplified using TransTaq HiFi DNA Polymerase (TransGen, Beijing, China) and cloned into the pET-28a vector using the ClonExpress MultiS One Step Cloning Kit (Vazyme, Nanjing, China). Primers were designed using PerlPrimer V1.1.21 and are listed in S2 Table. The linear vector pET-28a was generated by digestion with BamHI and XhoI (TaKaRa, Dalian, China). Recombinant plasmids were then transfected into E. coli BL21(DE3) Chemically Competent Cell (TransGen Biotech, Beijing, China) and confirmed by Sanger sequencing (Sangon Biotech, Shanghai, China). E. coli cells were grown in autoinduction medium [80 (link)] containing 100 μg/μl kanamycin at 300 rpm and 20°C for 48 h and then harvested by centrifugation at 12000 × g for 20 min. Recombinant protein was extracted using BugBuster Protein Extraction Reagent (Thermo Scientific, USA) and purified using cOmplete His-Tag Purification Resin (Roche, Switzerland) and His-Bind Purification kit (Novagen, USA) according to the manufacturer’s protocol. The concentration of purified protein was determined using a Pierce BCA Protein Assay Kit (Thermo Scientific, USA).

Fusions detected in our cohort were confirmed using fluorescence in situ hybridization (FISH) and reverse transcription–PCR (RT-PCR) followed by Sanger sequencing. FISH analyses were performed using dual-color probes EP300 and ZNF384 (Guangzhou LBP Medicine Science and Technology, China) according to the manufacturer’s instruction. The analyses were performed using a Zeiss Axioplan 2 microscope (Carl Zeiss AG, Oberkochen, Germany) and the CytoVision software (Leica Biosystems, Nussloch, Germany). For each patient, 200 interphase cells were analyzed.
For detection of the EP300-ZNF384 fusion transcript, RT-PCR was performed using PrimeScript^TM RT reagent Kit (Takara, Japan) and TransTaq® HiFi DNA Polymerase (Transgen, China). Two forms of EP300-ZNF384 fusion were amplified with the same forward primer (5′-AATCAGATGCCGACACAACC-3′) and reverse primer (5′-CAGCAAGGTGGGGTAGTGAG-3′). The resulting 321 bp and 261 bp amplicons were confirmed by Sanger sequencing (Sangon Biotech, China).

The amino acid sequence of D. melanogaster TMC protein was used to screen against N. lugens genomic and transcriptomic databases for identification of its homologs in N. lugens. Open reading frames (ORFs) were predicted with EditSeq (version 5.02, DNAstar, Madison, WI, United States).
Total RNA was isolated from whole insects using the TRIzol Reagent (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s protocol. Residual genomic DNA was removed by RQ1 RNase-Free DNase (Promega). Single-stranded cDNA was synthesized from the total RNA with M-MLV reverse transcriptase and oligo (dT)₁₈ (BioTeke, Beijing, China). The forward primer Nltmc-comp-F and the reverse primer Nltmc-comp-R were used to amplify the full-length or fragment gene by means of PCR on cDNA from adult N. lugens using TransTaq HiFi DNA Polymerase (TransGen Biotech, Beijing, China). The purified PCR products were sub-cloned into pGEM^®-T easy vector (Promega, Madison, WI) and then sequenced using the 3730 XL DNA analyzer (Applied Biosystems, Carlsbad, CA, United States. The primers corresponding to each gene are listed in Table 1.

3′-RACE was performed using 3′-Full RACE Core Set with PrimeScript RTase (TaKaRa) in accordance with manufacturer's instructions. Nested PCR was conducted in 3′-RACE outer primer and 3′-RACE-GSP1 or 3′-RACE inner primer and 3′-RACE-GSP2 (Table 1). The first round of PCR was performed using a reaction mixture containing 1 μL of the first-strand cDNA, 0.5 μL of each primer (10 μM), 2 μL of 10x Trans Taq HiFi buffer, 2 μL of dNTPs (2.5 mM), 0.3 μL of Trans Taq HiFi DNA Polymerase (TransGen Biotech), and 13.7 μL of MilliQ H₂O. The second round of PCR was conducted using a reaction mixture with 2 μL of outer PCR purified product, 1 μL of each primer (10 μM), 5 μL of 10x Trans Taq HiFi buffer, 4 μL of dNTPs (2.5 mM), 0.5 μL of Trans Taq HiFi DNA polymerase, and 36.5 μL of MiliQ H₂O. The amplifications of the first round were performed with initial denaturation at 94°C for 3 min, 30 cycles with denaturation at 94°C for 30 s, annealing at 48°C for 30 s, extension at 72°C for 50 s, and the final extension step at 72°C for 10 min. The second round was performed in the same manner as that of the first round except annealing at 56°C. The inner PCR product was ligated with pUM-T vector and further purified and transformed into DH5α. The detailing process was the same as above. The sequence was then determined (Nanjin Jinruisi Biotechnology Ltd., Co., China).