T4 dna polymerase

Manufactured by Qiagen

Sourced in United States

T4 DNA polymerase is a thermostable enzyme used in DNA replication and amplification. It catalyzes the polymerization of nucleotides into DNA strands in a template-dependent manner.

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Lab products found in correlation

T4 dna ligase, by Qiagen (3 mentions) Trans5α competent cell, by Transgene (2 mentions) Accuprep plasmid mini extraction kit, by Bioneer (2 mentions) Agencourt ampure kit, by Beckman Coulter (1 mentions) Sequencing library qpcr quantification guide, by Illumina (1 mentions) Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions) Kapa sybr fast universal qpcr kit, by Roche (1 mentions) Phix control v3, by Illumina (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions) Qubit dsdna br assay kit, by Thermo Fisher Scientific (1 mentions) Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions) Ampure bead, by Beckman Coulter (1 mentions) Qiaquick pcr purification kit, by Qiagen (1 mentions) Q5 polymerase, by New England Biolabs (1 mentions) Bioruptor, by Diagenode (1 mentions) T4 pnk, by Qiagen (1 mentions) Ez dna methylation kit, by Zymo Research (1 mentions) Dneasy mini kit, by Qiagen (1 mentions) Kapa hifi hotstart readymix, by Roche (1 mentions) E220 ultrasonicator, by Covaris (1 mentions)

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DNA polymerase (4 citations)

9 protocols using t4 dna polymerase

Illumina ChIP-Seq Library Preparation

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ChIP libraries were generated according to the Illumina ChIP-seq library construction procedure. Briefly, 10 ng of ChIP DNA was end repaired with T4 DNA polymerase, Klenow fragment and T4 polynucleotide kinase (all from Enzymatics), and an adenosine base was then added to the 3' end of the blunt phosphorylated DNA fragments by Klenow Fragment (3′→5′ exo-) (Enzymatics). This was followed by ligation of Illumina genomic adapters. Ligated DNA around the size of 250-300 bp was isolated by electrophoresis through a 3% NuSieve 3:1 agarose gel and amplified by PCR using Phusion DNA Polymerase (Thermo Scientific). The PCR products were then purified using an Agencourt Ampure kit (Beckman Coulter) to remove primer dimmers.
Agilent 2100 Bioanalyzer was employed to examine the normal size distribution and possible primer or linker contamination of each library. The library concentration was measured using both Qubit dsDNA BR Assay Kit (Invitrogen) and real-time qPCR method. Real-time qPCR was performed on StepOne Plus Real-Time PCR systems (Applied Biosystems) using KAPA SYBR FAST universal qPCR kit (KAPA Biosystems). qPCR primers, 1.1 and 2.1, were designed based on Illumina Sequencing Library qPCR Quantification Guide. PhiX Control v3 (Illumina) was used in the reaction to plot the standard curve.

Wang X., Yang L., Choi J.H., Kitamura E., Chang C.S., Ding J., Lee E.J., Cui H, & Ding H.F. (2014). Genome-wide analysis of HOXC9-induced neuronal differentiation of neuroblastoma cells. Genomics Data, 2, 50-52.

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Detailed ChIP Assay Protocol for hESCs

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For the ChIP experiments, 3 × 10⁶ hESCs were double-crosslinked with 0.2 mM di (N-succinimidyl) glutarate (DSG, Sigma, 80424) followed by 1% formaldehyde. The cell lysate was precleared and incubated with antibody overnight at 4°C. The DNA was purified using the Qiaquick PCR purification kit (QIAGEN, 28106). For ChIP-seq experiments, the ChIP DNA was end repaired and 5′ phosphorylated using T4 DNA Polymerase, Klenow, and T4 Polynucleotide Kinase (Enzymatics). Adaptor-ligated ChIP DNA fragments were subjected to 15 cycles of PCR amplification using Q5 polymerase (NEB). AMPure beads were used to purify DNA after each step (Beckman Coulter).

Estarás C., Benner C, & Jones K.A. (2015). SMADs and YAP Compete to Control Elongation of β-Catenin:LEF-1-Recruited RNAPII during hESC Differentiation. Molecular cell, 58(5), 780-793.

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Whole Genome Bisulfite Sequencing

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Genomic DNA (10 μg) was extracted from the embryo and endosperm using the DNeasy Mini Kit (Qiagen). The DNA was fragmented by sonication to 280–350 nt with a Bioruptor (Diagenode). The DNA was end-repaired using a mixture of T4 DNA polymerase, Klenow DNA polymerase and T4 PNK (Enzymatics), and a 3’ overhang A was added using the Klenow exo-enzyme (Enzymatics). The resultant fragments were ligated with the Illumina methylation adapters by DNA T₄ ligase (Enzymatics) according to the Illumina protocol. Adapter-linked DNA fragments were bisulfated using the EZ DNA Methylation Kit (Zymo), as per the manufacturer’s protocol. The treated DNA was amplified by PCR for 11 cycles. The DNA fragments were purified, quantified and then sequenced for 100 cycles using the Illumina protocol.

Lu X., Wang W., Ren W., Chai Z., Guo W., Chen R., Wang L., Zhao J., Lang Z., Fan Y., Zhao J, & Zhang C. (2015). Genome-Wide Epigenetic Regulation of Gene Transcription in Maize Seeds. PLoS ONE, 10(10), e0139582.

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Fungal DNA Extraction and Library Prep

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The fungi DNA was extracted using CTAB method and sheared into fragments between 100 and 500 bp in size by Covaris E220 ultrasonicator (Covaris, Brighton, UK). High-quality DNA were selected using AMPure XP beads (Agencourt, Beverly, MA, USA). After repairing using T4 DNA polymerase (Enzymatics, Beverly, MA, USA), the selected fragments were ligated at both ends to T-tailed adapters and amplified using KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Wilmington, NC, USA). Then amplification products were subjected to a single-strand circularization process using T4 DNA Ligase (Enzymatics) and generated a single-stranded circular DNA library.

Peng L., Li L., Liu X., Chen J., Shi C., Guo W., Xu Q., Fan G., Liu X, & Li D. (2019). Chromosome-Level Comprehensive Genome of Mangrove Sediment-Derived Fungus Penicillium variabile HXQ-H-1. Journal of Fungi, 6(1), 7.

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RNA-Seq Library Preparation from HAECs

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HAECs were resuspended in RNA Lysis Buffer and RNA was extracted from cells using the Quick-RNA Micro Prep kit from ZymoResearch (Irvine, CA, #R1051), including optional DNase I treatment. mRNA was selected through poly-A isolation using Oligo d(T)25 beads (New England BioLabs, Ipswich, MA, #S1419S). Selected RNA was fragmented, followed by single strand cDNA synthesis using a SuperScript III First-Strand Synthesis System (ThermoFisher Scientific # 18080051), followed by second strand synthesis using DNA Polymerase I (Qiagen/Enzymatics, Beverly, MA, #P7050L). dsDNA ends were repaired with T4 DNA Polymerase (Enzymatics #P7080L). Barcode adapters (BIOO Scientific NEXTflex, Austin, TX, #514104) were ligated onto the ends of sequences using T4 DNA Ligase (Enzymatics #L-6030-HC-L) and samples were treated with Uracil DNA Glycosylase (UDG) (Enzymatics #G5010L). Libraries were then amplified by PCR (Phusion Hot Start II, ThermoFisher Scientific, #F549L) and purified (Zymo #D5205) for high-throughput sequencing.

Hogan N.T., Whalen M.B., Stolze L.K., Hadeli N.K., Lam M.T., Springstead J.R., Glass C.K, & Romanoski C.E. (2017). Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells. eLife, 6, e22536.

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Hi-C sequencing of primate brain

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Frozen PFC samples (0.2 g) from a human and a macaque were ground in liquid nitrogen, after which cell suspensions were prepared. The filtered cell suspensions were fixed in 1% formaldehyde for 30 min. The cross-linked DNA was digested with 200 U MboI (NEB, #R0147M), and the digested fragment ends were filled with biotin-14-dATP (Invitrogen, #19524016), dCTP, dGTP, and dTTP by DNA Polymerase I Klenow Fragment (NEB, #M0210L). The resulting blunt-end fragments were religated by T4 DNA ligase (Enzymatics, #L603-HC-L). Then, the ligated cross-linked DNA was reversed using proteinase K (TIANGEN, #RT403). DNA purification was performed by removing biotin from unligated ends using T4 DNA polymerase (Enzymatics, #P7080L). The purified DNA was then sheared to a length of 350 to 500 bp using Covaris M220, and biotin-labeled DNA was pulled down with Dynabeads M-280 Streptavidin (Invitrogen, #11205D). Library preparation was performed using a DNA library preparation kit (Vazyme, #ND607). The libraries were then sequenced on the Illumina NovaSeq 6000 sequencing platform to generate 150-bp paired-end reads.

Ding W., Li X., Zhang J., Ji M., Zhang M., Zhong X., Cao Y., Liu X., Li C., Xiao C., Wang J., Li T., Yu Q., Mo F., Zhang B., Qi J., Yang J.C., Qi J., Tian L., Xu X., Peng Q., Zhou W.Z., Liu Z., Fu A., Zhang X., Zhang J.J., Sun Y., Hu B., An N.A., Zhang L, & Li C.Y. (2024). Adaptive functions of structural variants in human brain development. Science Advances, 10(14), eadl4600.

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Lentiviral sgRNA Vector Cloning

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10 μl of the sgRNA entry vector (200 ng/μL in H₂O) were digested with ApaI, SpeI, and EcoRV (FastDigest, Fermentas) at 37 °C for 2 h. The digestion mix was separated on an agarose gel (1%), the cleaved product was purified using an innuPrep gel extraction kit (Analytik Jena) and eluted in 15 μl H₂O (final concentration: 70 ng/μl). The linearized vector was chewed using T4 DNA polymerase (Enzymatics) for 5 min at 27 °C in the presence of dTTP as a stopping nucleotide. After heat-inactivation, the reaction was diluted 10-fold in 2× NEB2 buffer in the presence of 0.5 μM short universal reverse strand oligo (PAGE purified, 100 μM, IDT). Gene-specific sgRNA oligos (desalted, 100 μM, IDT) were diluted 1:400 in H₂O and then mixed at equal volume with the vector pre-dilution and annealed in a PCR cycler (C100, Biorad). For a detailed protocol please refer to the Supplementary Methods section.

Schmidt T., Schmid-Burgk J.L, & Hornung V. (2015). Synthesis of an arrayed sgRNA library targeting the human genome. Scientific Reports, 5, 14987.

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Ligation-Independent Cloning of VIGS Vectors

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The pTRV2-LIC vectors were constructed using the ligation independent cloning (LIC) method as previously described (Kim et al. 2017) . Partial coding sequences (200-400 bp) of the CaAN3 candidate gene were amplified with LIC adapter primers. The resulting purified PCR amplicon was treated with T4 DNA polymerase (Enzymatics, Beverly, MA, USA) with 1× blue buffer and 10 mM dATP. The pTRV2-LIC vector was digested with PstI and treated with T4 DNA polymerase in 1× buffer and 10 mM dTTP. T4 DNA polymerase-treated mixtures were incubated at 22ºC for 30 min, followed by 75ºC for 20 min. The two reaction products were then mixed in a 1:3 (vector:insert) ratio. For ligation, the mixture was incubated at room temperature for 30 min, then transformed into Trans5α competent cell (TransGen Biotech, Beijing, China). Plasmids were extracted using AccuPrep® Plasmid Mini Extraction Kit (Bioneer) and sequenced (Macrogen). Plasmids with complete sequences were introduced into Agrobacterium (Agrobacterium tumefaciens) strain GV3101 by electroporation. pTRV2::PDS and pTRV2::GFP were kindly provided by Prof. Doil Choi (Seoul National University).
Primers used for the VIGS study are listed in Table S1.

Byun J., Kim T.G., Lee J.H., Li N., Jung S, & Kang B.C. (2022). Identification of CaAN3 as a fruit-specific regulator of anthocyanin biosynthesis in pepper (Capsicum annuum). TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik, 135(7).

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Ligation-Independent Cloning of VIGS Vectors

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Byun J., Kim T., Lee J., Li N., Jung S., & Kang B. (2022). Identification of CaAN3 as a fruit-specific regulator of anthocyanin biosynthesis in pepper (Capsicum annuum).

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