In order to detect mtDNA in C6, C6ρ0 and cybrids, total DNA from a different number of cells was isolated as a template using a commercial kit (Tiangen Biotech Co., Ltd.), following the manufacturer's instructions. Oligonucleotide primers were used to amplify the nucleotide sequences of rat mtDNA mitochondrial D-loop region. The primers were synthesized by Sangon Biotech Co., Ltd. mtDNA was amplified using a volume of 50 µl containing 0.25 µl units of TakaRa Ex Taq polymerase (Takara Biotechnology Co., Ltd.) and 0.2 µl primer pair. Primers specific for the mtDNA D-loop region were the following: Forward 5′-CCTCCCATTCATTATCGCCGCCCTTGC-3′ and reverse 5′-GTCTGGGTCTCCTAGTAGGTCTGGGAA-3′ (235 bp). The PCR conditions were: 35 cycles of denaturation at 98°C for 10 sec, annealing at 60°C for 30 sec and extension at 72°C for 60 sec. The products were separated by 1% agarose gel electrophoresis and images were detected using a 5200 Multi Luminescent image analyzer, according to the manufacturer's protocol (Tanon Science and Technology Co., Ltd.). The intensity of the bands indicated the amount of mtDNA.
Takara ex taq polymerase
Manufactured by Takara Bio
Sourced in Japan, China, United States
TaKaRa Ex Taq polymerase is a high-performance DNA polymerase designed for PCR amplification. It exhibits robust enzymatic activity and high fidelity.
Automatically generated - may contain errors
Lab products found in correlation
High capacity rna to cdna kit, by Thermo Fisher Scientific (3 mentions)
Dntp mix, by Thermo Fisher Scientific (2 mentions)
Mvs 0066, by Maixin Group (2 mentions)
Sp kit a2, by Maixin Group (2 mentions)
Pbs 0060, by Maixin Group (2 mentions)
Kit 5004, by Maixin Group (2 mentions)
Permount mounting medium, by Thermo Fisher Scientific (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
3730xl automated sequencer, by Thermo Fisher Scientific (2 mentions)
Bigdye terminator v3.1 sequencing kit, by Thermo Fisher Scientific (2 mentions)
Applied biosystems 7900ht fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Oligo dt 18 primer, by Thermo Fisher Scientific (2 mentions)
Target specific primers, by Integrated DNA Technologies (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Thunderbird sybr qpcr mix, by Toyobo (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
35 protocols using takara ex taq polymerase
1
Quantifying Mitochondrial DNA in Cell Lines
2
16S rDNA V3 Region Amplification Protocol
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The 16S rDNA V3 region was amplified using a hot-start touchdown protocol with primers specific for conserved regions of the 16S rRNA gene [18 (link)]. The reaction mixture contained 2 μL of genomic DNA, 25 pmol of each primer, 4 μL of dNTPs, 5 μL of 10х Ex Taq buffer, 0.5 μL of TaKaRa Ex Taq polymerase (TaKaRa, Dalian, China), and sterile deionized water to a total volume of 50 μL. To minimize heteroduplex formation, five-cycle reconditioning PCR was conducted with 5 μL of amplification mixture in a fresh reaction mixture as previously described [19 (link)]. Profiles were generated using a TProfessional Thermocycler (Biometra, Göttingen, Germany). Amplified products were confirmed by agarose gel electrophoresis (1%), and their concentrations were measured using a NanoDrop ND-1000 spectrophotometer (Nano-Drop Technologies, Wilmington, DE, USA). All amplified products were stored at -20°C until DGGE analysis.
3
Bacterial Genotyping by Rep-PCR
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Genotyping of the isolates was performed by repetitive elements sequence-based PCR (Rep-PCR) with primers ERIC2 or BOXA1R (Table 6 ). The reaction mixture (20 μL) contained 2 μL of TaKaRa 10× Ex Taq Buffer, (Takara Biotechnology (Dalian) Co., Ltd., China), 2.0 mM MgCl2, 250 μM of each dNTP, 1.0 μM of one of the primers, 0.5 U of TaKaRa Ex Taq polymerase (Takara Biotechnology (Dalian) Co., Ltd., China) and 0.5 μL of a bacterial lysate as a template. Rep-PCRs were started by denaturation at 95 °C for 3 min. followed by 4 cycles at 94 °C for 1 min., 40 °C (ERIC-PCR) or 55 °C (BOX-PCR) for 1 min., 68 °C for 8 min.; followed by 30 cycles: 94 °C for 1 min., 52 °C (ERIC-PCR) or 65 °C (BOX-PCR) for 1 min., 72 °C for 2 min. A final extension was performed at 72 °C for 5 min.
Products of the amplification were separated by electrophoresis on a 2.0 % agarose gel (Genview, China) in 0.5 × TBE. The gel was supplemented with 50 μL L−1 GoldView Nucleic Acid Stain (Beijing Dingguochangsheng Biotechnology Co., Ltd., China) in order to visualize DNA bands. The Rep-PCR banding patterns were analyzed visually, and similar ones were considered to belong to the same genotypic group.
Products of the amplification were separated by electrophoresis on a 2.0 % agarose gel (Genview, China) in 0.5 × TBE. The gel was supplemented with 50 μL L−1 GoldView Nucleic Acid Stain (Beijing Dingguochangsheng Biotechnology Co., Ltd., China) in order to visualize DNA bands. The Rep-PCR banding patterns were analyzed visually, and similar ones were considered to belong to the same genotypic group.
4
Immunohistochemical Staining of AMHR2 and c-Kit
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The rabbit polyclonal antibodies used in this study were anti-AMHR2 antibody [MIG7] (ab64762, abcam, Shanghai, China) and anti-c-Kit antibody (ab115801, abcam, Shanghai, China). Other major chemicals and Kits were: TaKaRa Ex Taq Polymerase (DRR001A, TaKaRa Biotechnology [Dalian] Co. Ltd, Dalian, China), agarose gel (A600014-0050, Sangon Biotech, Shanghai, China), QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), Permount Mounting Medium (Thermo Fisher Scientific, Waltham, MA), dNTP mix (Life Technologies, Beijing, PR China), citrate-buffered solution (MVS-0066, MAIXIN Bio, Fuzhou, China), the endogenous peroxidases (SP KIT-A2, MAIXIN Bio, Fuzhou, China), phosphate-buffered saline (PBS-0060, MAIXIN Bio, Fuzhou, China), biotinylated goat anti-rabbit antibody (KIT-5004, MAIXIN Bio, Fuzhou, China), DAB (diaminobenzidine) Horseradish Peroxidase Color Development Kit (DAB-0031, MAIXIN Bio, Fuzhou, China), etc.
5
Transcriptomic Analysis of Insect Species
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Total RNA was extracted from Phyllodes eyndhovii from Lepidoptera, Panorpa vulgaris from Mecoptera, and Tunga penetrans from Siphonaptera as part of the 1KITE project. The RNA (2–5 µg) was reverse transcribed into cDNA using Oligo (dT)15 Primer and M-MLV reverse transcriptase (Promega, WI, USA) in a 50 µL reaction volume, with the reverse transcription mixture incubated at 42 °C for 1 h. The primers are listed in Table 1 . The PCR mixture (25 µL) consisted of 5 µL of cDNA template, 0.2 µM primers, 200 µM dNTP mix, and 2.5 U of TaKaRa Ex Taq polymerase (Takara, Shiga, Japan). The amplification conditions were as follows: initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, and extension at 72 °C for 30 s, with a final extension at 72 °C for 10 min. All PCR products were sequenced using Sanger sequencing (Ruibiotech, Beijing, China).
6
Validating Gene Fusion Transcripts
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To discover gene fusion from RNA-seq data, we used DeFuse version 0.4.3 and ChimeraScan version 0.4.59 (link)10 (link).
In order to validate fusion transcript by Sanger sequencing, fusion candidate were selected. Fusion transcripts were observed only in cancer tissues, and protein coding transcripts were selected. Genes that were reported in cancer gene database (COSMIC, ChimerDB 2.0) and previous studied were validated.
For Sanger sequencing, 2 µg of total RNA was used for cDNA synthesis with an oligo-dT primer and PrimeScript reverse transcription polymerase chain reaction Kit (Takara, Kyoto, Japan) according to the manufacturer's protocol.
Fusion gene specific primer pairs and TAKARA Ex-Taq polymerase (Takara) were used for the PCR reaction. After purification, PCR products were sequenced with the BigDye Terminator v3.1 Sequencing Kit and a 3730xl automated sequencer (Applied Biosystems, Foster City, CA, USA). All DNA sequenced comparison alignments were performed using DNAstar SeqMan program (DNAstar, Madison, WI, USA).
In order to validate fusion transcript by Sanger sequencing, fusion candidate were selected. Fusion transcripts were observed only in cancer tissues, and protein coding transcripts were selected. Genes that were reported in cancer gene database (COSMIC, ChimerDB 2.0) and previous studied were validated.
For Sanger sequencing, 2 µg of total RNA was used for cDNA synthesis with an oligo-dT primer and PrimeScript reverse transcription polymerase chain reaction Kit (Takara, Kyoto, Japan) according to the manufacturer's protocol.
Fusion gene specific primer pairs and TAKARA Ex-Taq polymerase (Takara) were used for the PCR reaction. After purification, PCR products were sequenced with the BigDye Terminator v3.1 Sequencing Kit and a 3730xl automated sequencer (Applied Biosystems, Foster City, CA, USA). All DNA sequenced comparison alignments were performed using DNAstar SeqMan program (DNAstar, Madison, WI, USA).
7
Random Mutagenesis of Cut7 Gene
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The cut7 ts mutants were constructed by PCR-based random mutagenesis. The GFP epitope tag and G418-resistance marker gene cassette (kanR) were first inserted into the 3′ flanking region of the cut7 gene (cut7-GFP-kanR). The cut7-GFP-kanR fragment purified from this strain was amplified/mutagenised with PCR using TaKaRa EX taq polymerase (Takara Bio Inc., Shiga, Japan) and transformed into a klp9∆ strain (deleted by hphR). G418 (and Hygromycin B)-resistant colonies were picked up and backcrossed with a wild type strain. After confirming co-segregation between G418/Hygromycin B-resistance and the ts phenotype, nucleotide sequencing was performed to determine the mutated sites within the cut7 gene of these mutants.
8
Intron Amplification by Gradient PCR
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To amplify each intron region, gradient PCR was used to optimize suitable conditions using an annealing temperature ranging between 50 and 60 °C. The PCR conditions were initial denaturation at 94 °C for 30 s, annealing at 50–60 °C for 30 s, extension at 72 °C for 1 min, and a final extension 72 °C for 5 min. In the case of low concentration (no/faint band), one microliter of the first PCR product was used as the DNA template for the second PCR using the same conditions as used with the first PCR. The PCR mixture contained 1× TaKaRa Ex PCR buffer, 0.2 mM dNTPs (each), 0.2 μM of each primer, and 1.0 U of TaKaRa Ex Taq polymerase (Takara Bio Inc., Shiga, Japan). Subsequently, the PCR product underwent electrophoresis in 1% agarose gel and was visualized with GelRedTM Nucleic Acid Gel Stain (Biotium, Inc., Hayward, CA, USA). The PCR product (~1000 bp) was cut for gel purification using an E.Z.N.A.® Gel Extraction kit (Omega bio-tek, Norcross, GA, USA) and was subsequently used for DNA sequencing and cloning.
9
Genetic Sequencing of MYH7 and HSPA6 Genes
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Genomic DNA was extracted from whole peripheral blood leukocytes. The MYH7 and HSPA6 genes were amplified by using TaKaRa Ex Taq polymerase (Takara Bio, Shiga, Japan) and KOD Plus Ver. 2 DNA polymerase (TOYOBO, Osaka, Japan), respectively, according to the corresponding manufacturers’ instructions. PCR products were direct-sequenced on the 3730xl DNA analyzer (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA) after purification by Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). Primer sequences for PCR and sequencing of the MYH7 and HSPA6 genes are available on request.
10
EGFR T790M Mutation Detection
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Quantitative real-time PCR and EGFR mutation analysis was done as previously described (13 (link), 25 (link)). All experiments were performed in a triplicate assays. To analyze the T790M mutation, exon 20 of the EGFR gene was amplified using the PCR primer set and TaKaRa Ex Taq polymerase (TaKaRa BIO, Inc). PCR products were directly used as templates for cycle sequencing reactions using the BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems). The forward or reverse primers were used for cycle sequencing reactions, which were carried out in an ABI PRISM 310 Genetic Analyzer.
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