Deoxynucleotide triphosphate

Manufactured by New England Biolabs

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Deoxynucleotide triphosphates (dNTPs) are the building blocks of DNA. They are the four different nucleotides (dATP, dGTP, dCTP, and dTTP) that are used by DNA polymerase to synthesize new DNA strands during DNA replication and repair processes.

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Lab products found in correlation

Specific primers, by Macrogen (1 mentions) Dnazol reagent, by Thermo Fisher Scientific (1 mentions) Taq dna polymerase, by Thermo Fisher Scientific (1 mentions) Mycycler, by Bio-Rad (1 mentions) Mgcl2, by Thermo Fisher Scientific (1 mentions) Isothermal amplification buffer, by New England Biolabs (1 mentions) Bst 2.0 dna polymerase, by New England Biolabs (1 mentions) Warmstart rtx reverse transcriptase, by New England Biolabs (1 mentions) Mgso4, by New England Biolabs (1 mentions) Syto9, by Thermo Fisher Scientific (1 mentions) Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions) T4 dna polymerase, by Takara Bio (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions)

Close spelling variants of this product

Deoxynucleotides (4 citations) Deoxynucleotide triphosphates (dNTPs) (4 citations) Four deoxynucleotide triphosphates (dNTPs) (2 citations) Deoxynucleotide triphosphate mix (2 citations) Ultrapure equimolar deoxynucleotide triphosphate (dNTP) sodium salt solution (2 citations)

4 protocols using deoxynucleotide triphosphate

Genomic DNA Extraction and PCR Amplification

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T. brucei brucei Lister 427 PCF collected by centrifugation were washed twice with PBS (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.4) and genomic DNA was isolated using DNAzol Reagent as described by the manufacturer (Life Technologies, Carlsbad, CA, USA).
Polymerase Chain Reaction (PCR) was performed at a final volume of 50 μl containing genomic (~300 ng) or plasmid DNA (~50 ng), 30 pmoles of the specific primers (Macrogen, Seoul, Korea), 2.5 mM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP) (New England Biolabs), 1.5 mM MgCl₂ (Life Technologies), 0.2 UI Taq DNA polymerase (Life Technologies) and reaction buffer as indicated by the manufacturer (Life Technologies).
For the amplification reaction a thermocycler (Mycycler, Bio-Rad, Hercules, CA, USA) was employed: an initial denaturation step at 94°C for 10 min was followed by 35 cycles of a) denaturation at 94°C 1 min, b) hybridization at 55°C 1 min, c) elongation at 72°C -1 min per kb of DNA amplified; the final elongation step was performed at 72°C for 10 min.

Iribarren P.A., Berazategui M.A., Cazzulo J.J, & Alvarez V.E. (2015). Biosynthesis of SUMOylated Proteins in Bacteria Using the Trypanosoma brucei Enzymatic System. PLoS ONE, 10(8), e0134950.

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Amplification of nifH Genes Using Modified Primers

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PCR amplification was carried out using 4 U Taq DNA polymerase in 1× Taq buffer (containing 1.5 mM MgCl₂) (Qaigen, Valencia, CA, USA) in a 100 µL reaction mixture containing 25 ng of DNA template, 800 µM of each deoxynucleotide triphosphate (New England Biolabs, Ipswich, MA, USA), 0.5 pmol µL⁻¹ of each primer (MWG Biolabs, Huntsville, AL, USA) and 40 mg bovine serum albumin (NEB). The nifH primers used (Forward primer, nifH F; Reverse primer, nifH R) [24 (link),54 (link)] contain the artificial nucleotides P [55 (link)] and K [56 (link)]. The use of these artificial nucleotides reduces degeneracy and occurrence of spurious products. PCR amplification was performed using the program described by Davis et al. [27 (link)]. Ninety microliters of the reaction mixture were concentrated by isopropanol precipitation. Amplicons were recovered by centrifugation for 30 min at 10,000× g, washing in 70% ethanol and recovery in 10 μL of TE (pH 8.0; 10 mM Tris-HCl, 1 mM EDTA).

Davis D.A., Malone S.L, & Lovell C.R. (2018). Responses of Salt Marsh Plant Rhizosphere Diazotroph Assemblages to Drought. Microorganisms, 6(1), 27.

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RT-LAMP for Influenza HA Detection

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RT-LAMP was performed as described in the previous studies with some modifications [12 (link),13 (link),15 (link),16 (link)]. In brief, 20 μL of LAMP reaction mixture containing 2 μL of 10× isothermal amplification buffer (New England Biolabs, Ipswich, MA, USA), 1.5 mM concentrations of each deoxynucleotide triphosphate (New England Biolabs), 6 mM MgSO₄ (New England Biolabs), 8 U of Bst 2.0 DNA polymerase (New England Biolabs), 7.5 U of WarmStart RTx Reverse Transcriptase (New England Biolabs), 1.25 μM SYTO9 (Thermo Fisher Scientific, Waltham, MA, USA), and 2 μL of template plasmid DNA of H1-HA or H3-HA was used. The composition of the RT-LAMP primer mix was 0.2 μM of the outer primers (F3 and B3), 1.6 μM of the inner primers (FIP and BIP), and 0.4 μM of the loop primers (LF and LB). The amplification reaction was performed at 60℃ for 45 min and terminated by heating at 80℃ for 3 min in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The fluorescence curve was captured from the Bio-Rad CFX Manager graph.

Kang J.S., Seo M.R, & Chung Y.J. (2022). Development of reverse-transcription loop-mediated isothermal amplification assays for point-of-care testing of human influenza virus subtypes H1N1 and H3N2. Genomics & Informatics, 20(4), e46.

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Construction of 8BrG-Containing pMY189 Shuttle Vector

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A shuttle vector pMY189, which contains the bacterial suppressor tRNA (supF) gene [23 (link)], was used for the construction of pMY189 containing a single 8BrG:cytosine pair. First, E. coli XL1-Blue MRF' (Stratagene, La Jolla, CA, USA) and R408 Helper Phage (Stratagene) were used to prepare single-stranded pMY189 DNA, and 30 μg of the single-stranded plasmid pMY189 and a 5-fold molar excess of 5′-phosphorylated 24-mer oligonucleotide with a single 8BrG at nucleotide position 159 of the supF gene [5′-CGA CTT CGA A(8BrG)G TTC GAA TCC TTC-3′] (Japan Bio Services, Saitama, Japan) were annealed in a reaction mixture. Forty units of T4 DNA polymerase (Takara, Kyoto, Japan), 600 μM of deoxynucleotide triphosphate, 2400 units of T4 DNA ligase (New England Biolabs, Beverly, MA, USA), and 1 mM of ATP were added to the reaction mixture, and the mixture was incubated at 37°C for 4 h. Then, closed circular pMY189 containing an 8BrG was isolated using cesium chloride-ethidium bromide density gradient centrifugation. To prepare wild-type pMY189, an oligonucleotide without modified bases [5′-CGA CTT CGA AGG TTC GAA TCC TTC-3′] was used, and a closed circular wild-type pMY189 was obtained in the same manner.

Shinmura K., Kato H., Goto M., Tao H., Inoue Y., Nakamura S., Yoshida H., Tsuzaki E, & Sugimura H. (2017). Mutation Spectrum Induced by 8-Bromoguanine, a Base Damaged by Reactive Brominating Species, in Human Cells. Oxidative Medicine and Cellular Longevity, 2017, 7308501.

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