Q5 high fidelity dna polymerase

Manufactured by Takara Bio

Sourced in China, United States

The Q5 High-Fidelity DNA Polymerase is a thermostable DNA polymerase enzyme that exhibits high accuracy and processivity for DNA amplification. It possesses 3' to 5' exonuclease activity, which provides proofreading capability to ensure high-fidelity DNA synthesis.

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Lab products found in correlation

Sd leu trp, by Takara Bio (1 mentions) Pgadt7, by Takara Bio (1 mentions) In fusion hd cloning, by Takara Bio (1 mentions) In fusion cloning, by Takara Bio (1 mentions) Nanoluc luciferase reporter plasmid, by Promega (1 mentions) Miseq system, by Illumina (1 mentions) Reaction buffer, by Takara Bio (1 mentions) Axygen axyprep dna gel extraction kit, by Corning (1 mentions) High throughput sequencing, by Illumina (1 mentions)

Spelling variants of this product

DNA polymerase (100 citations) High-fidelity DNA polymerase (17 citations) Q5 DNA polymerase (4 citations)

5 protocols using q5 high fidelity dna polymerase

Yeast Two-Hybrid and Split Luciferase Assays

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The coding sequence of FvGPA1 (FVEG_06962), FvGPA2 (FVEG_04170), FvGPA3 (FVEG_02792), FvGPB1(FVEG_05349), FvGBB1 (FVEG_10291), FvMKK1/2 (FVEG_05280), FvSLT2 (FVEG_03043), and FvBCK1(FVEG_05000) were used to construct plasmids for yeast two-hybrid assay. These were amplified from F. verticillioides strain M3125 cDNA using Q5® High-Fidelity DNA Polymerase and inserted in pGADT7 (Clontech, Mountain View, CA, USA) as prey vectors. The full length of the coding region of FvGbb2 was cloned into pGBKT7 as a bait vector. The resulting plasmids were verified by sequencing. The pair of yeast two-hybrid plasmids were transformed into yeast strain AH109 following the instruction. We added 5 μl of transformants (10 7 /mL) on SD/-Leu/-Trp (Clontech, Mountain View, CA, USA) and SD/-Ade/-His/-Leu/-Trp (3 mM 3-AT) agar plates.
Split luciferase complementation assay plasmids in this study were obtained as described previously (Kim et al., 2012) . The coding region of each gene was amplified from WT strain cDNA by Q5® High-Fidelity DNA Polymerase and inserted into pFNLucG or pFCLucH via In-Fusion® HD Cloning (Clontech, Mountain View, CA, USA). The resulting constructs were validated by sequencing. Transformation, selection and luciferin assay were followed by a previous description (Zhang et al., 2018b) .

Yan H, & Shim W.B. (2020). Characterization of non-canonical G beta-like protein FvGbb2 and its relationship with heterotrimeric G proteins in Fusarium verticillioides. Environmental microbiology, 22(2).

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JUN and HBB 5' UTR Nluc Reporters

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To generate the JUN 5′ UTR and the HBB 5′ UTR Nluc reporter plasmids, the JUN 5′ UTR (ENST00000371222.4) previously generated by amplification from human cDNA [18 (link)] and the HBB 5′ UTR (ENST00000335295.4) commercially generated (IDT) sequences were each inserted into the pNL1.1 NanoLuc luciferase reporter plasmid (Promega, GenBank Accession Number JQ437370) downstream of a T7 promoter using overlap-extension PCR with Q5 High-Fidelity DNA Polymerase (NEB) and InFusion cloning (Takara Bio). For the JUN AUG mutants, the first 51 nucleotides of the JUN CDS were inserted downstream of the full JUN 5′ UTR sequence and upstream of the full Nluc CDS in the pNL1.1 plasmid. For the Fluc reporter plasmid, the HBB 5′ UTR Nluc reporter plasmid was amplified and the NanoLuc luciferase sequence was replaced by a commercially generated Firefly luciferase sequence (IDT) [71 (link)]. Subsequent mutant versions of the JUN reporter plasmids were made by amplifying the plasmid using overlap-extension PCR with Q5 High-Fidelity DNA Polymerase (NEB) and primers containing the corresponding mutations, insertions, or deletions, followed by InFusion cloning (Takara). All primers used for amplification can be found in S2 Table. All sequences were verified by Sanger sequencing. Protocol available: http://dx.doi.org/10.17504/protocols.io.kqdg3xoyzg25/v1.[PROTOCOL DOI].

González-Sánchez A.M., Castellanos-Silva E.A., Díaz-Figueroa G, & Cate J.H. (2024). JUN mRNA translation regulation is mediated by multiple 5’ UTR and start codon features. PLOS ONE, 19(3), e0299779.

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Amplification and Sequencing of Gut Microbiome

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The DNA isolated from fecal samples was used as the template for the ampliﬁcation of the 16S rRNA V3-V4 region. The universal primers used were F (5’-NNNNNNN ACTCCTACGGGAGGCAGCA-3’) and R (5’-NNNNNNN GGACTACVSGGGTATCTAAT-3’), with the NNNNNNN being unique seven-base barcode used to tag each PCR product. The PCR reaction was performed according to the touchdown protocol[18 (link)] in a system of 25 μL containing 5.0 μL 5 × reaction buffer (TaKaRa, Dalian, China), 5.0 μL 5 × high GC buffer (TaKaRa, Dalian, China), 0.5 μL dNTPs (10 mmol/L) mixture , 1.0 µL forward primer (10 µmol/L), 1.0 μL reverse primer (10 µmol/L), 0.25 μL Q5 high-fidelity DNA polymerase (5 U/uL, TaKaRa, Dalian, China), and 1 μL DNA template. Each PCR product was puriﬁed by 2% agarose gel electrophoresis. DNA was isolated using the Axygen Axy Prep DNA Gel Extraction kit (Axygen, Shanghai, China). The sequencing was finished with the help of the Illumina Miseq System (Illumina).

Zhang J., Ren F.G., Liu P., Zhang H.K., Zhu H.Y., Feng Z., Zhang X.F., Wang B., Liu X.M., Zhang X.G., Wu R.Q, & Lv Y. (2017). Characteristics of fecal microbial communities in patients with non-anastomotic biliary strictures after liver transplantation. World Journal of Gastroenterology, 23(46), 8217-8226.

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Targeted DNA Amplification and Sequencing

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Targeted sites were amplified from the extracted DNA with the corresponding site-specific primers by using Q5 High-Fidelity DNA Polymerase (Takara, Beijing, China). In this experiment, we used two deep-sequencing platforms for on-target analysis. The identification primer (18–20 nt) was designed in accordance with the principles of conventional PCR primer design. Specifically, the detection site must be within the range of 10–100 bp in the forward or reverse primer, and the bridging sequence 5′–ggagtgagtacggtgtgc–3′ should be added to the front of the forward primer. At the same time, the bridging sequence 5′–gagttggatgctggatgg–3′ was added to the front end of the reverse primer. Each PCR was performed with a 30 μL volume comprising 2 μL of the template in accordance with the manufacturer’s protocol. The paired-end sequencing of PCR amplicons was performed by Illumina MiSeq.

Cheng L., Zhou X., Zheng Y., Tang C., Liu Y., Zheng S., Liu Y., Zhou J., Li C., Chen M., Lai L, & Zou Q. (2021). Simple and Rapid Assembly of TALE Modules Based on the Degeneracy of the Codons and Trimer Repeats. Genes, 12(11), 1761.

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16S rDNA Profiling of Leafhopper Gut Microbiome

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Genomic DNA of the gut microbiome of leafhoppers was amplified by primers V3-V4F (5′-ACTCCTACGGGAGGCA GCA-3′) and V3-V4R (5′-GGACTACHVGGGTWTCT AAT-3′) specific for the 16S rDNA hypervariable V3-V4 region. The PCR mix contained 1.5 μl PrimerF, 1.5 μl PrimerR, 0.5 μl Q5 High-Fidelity DNA Polymerase (TaKaRa, Dalian, China), 10 μl High GC Enhancer (TaKaRa), 10 μl 5 × PCR Buffer (TaKaRa), 1 μl dNTP (TaKaRa) and 40ng DNA and ddH₂O. The thermal cycling conditions for the indexing PCR consisted of an initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 1 min, 55°C for 1 min and 72°C for 1 min. The PCR products were purified, quantified and amplified again as template by Solexa PCR with an initial denaturation of 98°C for 30 s, followed by 40 cycles of 98°C for 10 s, 65°C for 30 s and 65°C for 30 s. Solexa PCR products were purified and quantified for high-throughput sequencing (Illumina) by Biomarker Technologies (Beijing, China).

Wang H., Wu N., Liu Y., Kundu J.K., Liu W, & Wang X. (2019). Higher Bacterial Diversity of Gut Microbiota in Different Natural Populations of Leafhopper Vector Does Not Influence WDV Transmission. Frontiers in Microbiology, 10, 1144.

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