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T3 dna ligase

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T3 DNA ligase is an enzyme used for the ligation of DNA fragments. It catalyzes the formation of phosphodiester bonds between the 3'-hydroxyl and 5'-phosphate ends of double-stranded or single-stranded DNA.

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Lab products found in correlation

T4 rna ligase buffer, by New England Biolabs (3 mentions) Myone streptavidin c1 magnetic beads, by Thermo Fisher Scientific (3 mentions) Dynabeads myone c1, by Thermo Fisher Scientific (2 mentions) Aminolink plus micro immobilization kit, by Thermo Fisher Scientific (2 mentions) Pcsk9 pc9 h5223, by ACROBiosystems (2 mentions) Il 6 il6 h4218, by ACROBiosystems (2 mentions) Hbs p, by GE Healthcare (2 mentions) T4 dna ligase, by New England Biolabs (2 mentions) T7 dna ligase, by New England Biolabs (2 mentions) T4 rna ligase reaction buffer, by New England Biolabs (2 mentions) Triton x 100, by Merck Group (1 mentions) Tris hcl, by Avantor (1 mentions) Pbcv 1 dna ligase, by New England Biolabs (1 mentions) Hbs p buffer, by GE Healthcare (1 mentions) Phosphoramidites, by Glen Research (1 mentions) Ethanol, by Sinopharm (1 mentions) Taq dna ligase, by New England Biolabs (1 mentions) Trichloromethane, by Sinopharm (1 mentions) T4 rna ligase 2, by New England Biolabs (1 mentions) Jumpstart taq dna polymerase, by Merck Group (1 mentions)

Close spelling variants of this product

DNA ligase (62 citations) Ligases (5 citations)

9 protocols using t3 dna ligase

1

Protein Interaction Assay Protocols

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Oligonucleotides were purchased from IDT, and are listed in the Supplementary Information. Monomer building blocks were synthesized as described in ref 34 . T3 DNA ligase (3,000 U/µL), ATP, T4 RNA ligase buffer were purchased from New England Biolabs. PCSK9 (PC9-H5223) and IL-6 (IL6-H4218) were purchased from AcroBiosystems and reconstituted according to the manufacturers recommendations. Aliquots were flash frozen in liquid N2 and stored at −80 ºC until ready to use. The proteins were prepared for SPR by dialyzing into the indicated buffer and concentration was determined by absorbance at 280 nm. Streptavidin-coated magnetic beads (Dynabeads MyOne C1) were purchased from Life Technologies. AminoLink Plus Micro Immobilization Kits (ThermoFisher Scientific) were used for protein immobilization and for blank beads. HBS-P+ was purchased from GE Healthcare Life Sciences.
Lichtor P.A., Chen Z., Elowe N.H., Chen J.C, & Liu D.R. (2019). Side-chain determinants of biopolymer function during selection and replication. Nature chemical biology, 15(4), 419-426.
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2

Single-channel Nanopore Protein Sensing

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The lipid bilayer was formed by 1,2-diphytanoyl-sn-glycero-3-phospho-choline (DPhPC) dissolved in decane at 10 mg/mL. The wild-type α-HL monomer was purchased from List Biological Laboratories as a lyophilized powder and dissolved in water at 0.2 mg/mL. Buffer A (100 mM KCl, 66 mM Tris·HCl, 7.5% PEG, 5 mM MgCl2, 1 mM ATP, at pH 7.6) was placed outside the capillary, which was the cis site of α-HL. Buffer B (100 mM KCl 66 mM Tris-HCl at pH 7.6) was used to fill the capillary to avoid the PEG molecule blocking the outlet of the DNA molecules. T3-DNA ligase at 3 × 106 units/mL and its cofactor ATP were purchased from New England Biolabs, Ipswich, MA. The volume excluder polyethylene glycol 6000 (PEG 6000) was purchase from Sigma.
Tan C.S., Riedl J., Fleming A.M., Burrows C.J, & White H.S. (2016). Kinetics of T3-DNA Ligase-Catalyzed Phosphodiester Bond Formation Measured using the α-Hemolysin Nanopore. ACS nano, 10(12), 11127-11135.
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3

Thermostable DNA Ligase Assay Protocol

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Thermus thermophilus (Tth) DNA ligase (sold under the name Thermus aquaticus (Taq) DNA ligase by New England Biolabs for historical reasons), PBCV-1 DNA ligase (sold as SplintR ligase by New England Biolabs), T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, 2 M KCl, 1 M MgCl2, 1 M DTT, 10 mM ATP, and 50 mM NAD+ were obtained from New England Biolabs (Ipswich, MA). Tris-HCl (1 M pH 7.5 @ 25°C) was obtained from Amresco (Solon, OH). Triton X-100 (10%) was obtained from Sigma-Aldrich (St Louis, MO). Tth DNA ligase buffer (20 mM Tris-HCl pH 7.5 @ 25°C. 25 mM KCl, 10 mM MgCl2, 1 mM NAD+, 10 mM DTT, and 0.1% Triton® X-100) was prepared as a 10X stock. T4/PBCV-1 DNA ligase buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP, 10 mM MgCl2, and 10 mM DTT) was prepared as a 5X stock. Oligonucleotide annealing buffer (10 mM Tris pH 7.5 @ 25°C, 50 mM KCl, 0.1 mM EDTA) was prepared as a 10X stock. Ligase reaction quench (50 mM EDTA, 0.1% Triton® X-100) was prepared at 1X.
Bauer R.J., Evans TC J.r, & Lohman G.J. (2016). The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase. PLoS ONE, 11(3), e0150802.
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4

Enzymatic Synthesis of Customized DNA

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DNA template [up to 10 pmol, either in solution or immobilized on MyOne Streptavidin C1 magnetic beads (ThermoFisher Scientific)], polymerization initiation and termination primers (1.5 equivalents each relative to template), functionalized trinucleotide building blocks (10 equivalents relative to template for each occurrence of the corresponding codon) and 10x T4 RNA ligase reaction buffer (New England Biolabs; 1 μL) were mixed in a total volume of 8 μL in a PCR tube. The mixture was subjected to the following temperature program on a thermocycler: 95 °C for 10 sec; 65 °C for 4 min; a ramp from 65 °C to 4 °C at 0.1 °C per 10 s. To the PCR tube were added 1 μL of 10 mM ATP and 1 μL of T3 DNA ligase (New England Biolabs). The reaction was incubated at 4 °C for 12 h and then at 16 °C for 2 h.
Chen Z., Lichtor P.A., Berliner A.P., Chen J.C, & Liu D.R. (2018). Evolution of Sequence-Defined Highly Functionalized Nucleic Acid Polymers. Nature chemistry, 10(4), 420-427.
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5

Enzymatic Synthesis of Customized DNA

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DNA template [up to 10 pmol, either in solution or immobilized on MyOne Streptavidin C1 magnetic beads (ThermoFisher Scientific)], polymerization initiation and termination primers (1.5 equivalents each relative to template), functionalized trinucleotide building blocks (10 equivalents relative to template for each occurrence of the corresponding codon) and 10x T4 RNA ligase reaction buffer (New England Biolabs; 1 μL) were mixed in a total volume of 8 μL in a PCR tube. The mixture was subjected to the following temperature program on a thermocycler: 95 °C for 10 sec; 65 °C for 4 min; a ramp from 65 °C to 4 °C at 0.1 °C per 10 s. To the PCR tube were added 1 μL of 10 mM ATP and 1 μL of T3 DNA ligase (New England Biolabs). The reaction was incubated at 4 °C for 12 h and then at 16 °C for 2 h.
Chen Z., Lichtor P.A., Berliner A.P., Chen J.C, & Liu D.R. (2018). Evolution of Sequence-Defined Highly Functionalized Nucleic Acid Polymers. Nature chemistry, 10(4), 420-427.
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6

Protein Interaction Assay Protocols

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Oligonucleotides were purchased from IDT, and are listed in the Supplementary Information. Monomer building blocks were synthesized as described in ref 34 . T3 DNA ligase (3,000 U/µL), ATP, T4 RNA ligase buffer were purchased from New England Biolabs. PCSK9 (PC9-H5223) and IL-6 (IL6-H4218) were purchased from AcroBiosystems and reconstituted according to the manufacturers recommendations. Aliquots were flash frozen in liquid N2 and stored at −80 ºC until ready to use. The proteins were prepared for SPR by dialyzing into the indicated buffer and concentration was determined by absorbance at 280 nm. Streptavidin-coated magnetic beads (Dynabeads MyOne C1) were purchased from Life Technologies. AminoLink Plus Micro Immobilization Kits (ThermoFisher Scientific) were used for protein immobilization and for blank beads. HBS-P+ was purchased from GE Healthcare Life Sciences.
Lichtor P.A., Chen Z., Elowe N.H., Chen J.C, & Liu D.R. (2019). Side-chain determinants of biopolymer function during selection and replication. Nature chemical biology, 15(4), 419-426.
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7

Oligonucleotide Synthesis and Immobilization

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Oligonucleotides were purchased from IDT and are listed in the Supplementary Information. Building blocks were synthesized as described previously16 ,21 (link). Building blocks were synthesized either directly from phosphoramidites (Glen Research) or via coupling of an amine to the NHS ester containing phosphoramidite as described previously16 ,21 (link). T3 DNA ligase, ATP, and T4 RNA ligase buffer were purchased from New England BioLabs. MyOne Streptavidin C1 magnetic beads (Life Technologies) were used in immobilization. MST samples were prepared using HBS-P + buffer (GE Healthcare Life Sciences).
Chen J.C., Chen J.P., Shen M.W., Wornow M., Bae M., Yeh W.H., Hsu A, & Liu D.R. (2022). Generating experimentally unrelated target molecule-binding highly functionalized nucleic-acid polymers using machine learning. Nature Communications, 13, 4541.
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8

Synthesis and Purification of RNA Segments

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RNA2577-A, RNA2577-m6A and RNA2488-A segments were synthesized by Integrated DNA Technologies (America). DEPC-treated water, RNAiso (lysis buffer), dNTPs and the DNA probes were obtained from TaKaRa Biotechnology Co. Ltd. (Dalian, China). JumpStart™ Taq DNA Polymerase was purchased from Sigma-Aldrich (Shanghai, China). T3 DNA ligase, T7 DNA ligase, T4 DNA ligase and Taq DNA ligase as well as T4 RNA ligase 2 were purchased from New England Biolabs (Beijing, China). Super Green I was purchased from Fanbo Biochemicals Co. Ltd. (Beijing, China). Trichloromethane, isopropyl alcohol and ethanol were purchased from Sinopharm Chemical Reagent Co. Ltd. (Beijing, China). All the solutions for reactions were prepared with DEPC-treated water.
Liu W., Yan J., Zhang Z., Pian H., Liu C, & Li Z. (2018). Identification of a selective DNA ligase for accurate recognition and ultrasensitive quantification of N6-methyladenosine in RNA at one-nucleotide resolution †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc05233b. Chemical Science, 9(13), 3354-3359.
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9

RNA Ligation Efficiency Assay

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A total of 300 ng total RNA was incubated with 20 nM synthesized Probe R and Probe L in the 7-μl reaction mixture of T3 DNA ligation buffer at 85 °C for 3 min and then 35 °C for 10 min (Table S4). 50U T3 DNA ligase (New England Biolabs) with an appropriate amount of ligation buffer was added into the RNA mixtures to the final volume of 10 μl. This mixture was incubated at 35 °C for 10 min, chilled on ice immediately, and then amplified by PCR. The amount of PCR products which could be used to assess the ligation efficiency was measured by agarose gel electrophoresis and RT-qPCR.
Liu B., Lv Y., Hu W., Huang Y., Ying X., Chen C., Zhang H, & Ji W. (2024). m6A modification mediates SLC3A2/SLC7A5 translation in 3-methylcholanthrene-induced uroepithelial transformation. Cell Biology and Toxicology, 40(1), 5.
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