Journal:

Article Title: Synapsis of Recombination Signal Sequences Located in cis and DNA Underwinding in V(D)J Recombination

doi: 10.1128/MCB.24.19.8727-8744.2004

Figure Lengend Snippet: Kinetics of RAG-mediated synapsis of cis RSSs. Complete ligation reactions were performed by adding a mixture containing 200 U of T4 DNA ligase, 12.5 nM GST-RAG2, and 6 nM MBP-RAG1-D708A to a solution containing 3 nM IS95 DNA in the presence of 2.5 mM

Article Snippet: T4 DNA ligase was then added, and all subsequent steps were performed as described above.

Techniques: Ligation

Journal: PLoS ONE

Article Title: MDP: A Deinococcus Mn2+-Decapeptide Complex Protects Mice from Ionizing Radiation

doi: 10.1371/journal.pone.0160575

Figure Lengend Snippet: Radioprotection of T4 DNA ligase by rationally-designed peptides. T4 DNA ligase was irradiated in the indicated mixtures and then tested for residual activity by incubation with linearized (L) pUC19 DNA (2.686 kbp). Formation of supercoiled (SC) and open-circular (OC) DNA forms were determined by agarose gel electrophoresis. Peptides were added to 25 mM Pi with or without 1 mM MnCl 2 to the following concentrations: DP1-L, 3 mM; DP1-D, 3 mM; DP2, 3 mM; OP1, 3.75 mM; HP1, 5 mM. Note, the final concentration of peptides in the reaction mixtures corresponded to 30 mM of total amino acid residues. Other abbreviations: Pi, potassium phosphate buffer (pH 7.4); M, DNA size standards (Mass Ruler DNA Ladder mix; Fermentas, Glen Burnie, MD).

Article Snippet: T4 DNA ligase T4 DNA ligase (2,000 U/μl) (New England Biolabs, Ipswich, MA) was diluted in the various reagent-mixtures to 2 U/μl for irradiation.

Techniques: Irradiation, Activity Assay, Incubation, Agarose Gel Electrophoresis, Concentration Assay

Journal: Plant Methods

Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

doi: 10.1186/s13007-018-0359-7

Figure Lengend Snippet: Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

Article Snippet: Standard restriction enzymes are sufficient for Pyrite cloning, but they should be tested for functionality and fidelity in the T4 DNA ligase buffer.

Techniques: Clone Assay, Plasmid Preparation, Purification, Polymerase Chain Reaction, Incubation, Transformation Assay

Journal: Nucleic Acids Research

Article Title: Quantitative disclosure of DNA knot chirality by high-resolution 2D-gel electrophoresis

doi: 10.1093/nar/gkz015

Figure Lengend Snippet: Test of the electrophoresis procedure that discerns DNA knot chirality. ( A ) A linear 4.4-kb DNA fragment was circularized in free solution with T4 DNA ligase to produce a small fraction of molecules containing a trefoil knot. Negative supercoils were subsequently introduced by incubating the circularized DNA with topoisomerase I in presence of 250 μg/ml chloroquine. ( B ) The gel-blot shows the DNA products after high resolution 2D-gel electrophoresis carried out in 0.9% agarose gel (40 × 23 cm) in TBE. The first gel dimension (top to bottom) was run at 80 V for 70 h in TBE (89 mM Tris-borate, pH 8.3, 2 mM EDTA). The second gel dimension (left to right) was run at 120 V for 10 h in TBE containing 0.65 μg/ml of chloroquine. Lk, linking number topoisomers. N, nicked unknotted circles. L, linear DNA. The enlarged gel section shows the signal of Lk topoisomers of unknotted molecules (Kn# 0) and of molecules containing either a positive- or negative-noded trefoil knot (Kn# 3). ( C ) Probability of the two chiral forms of the trefoil knot.

Article Snippet: The same DNA products were obtained by nicking the DNA with Nt-Bst NBI endonuclease (NEB) and sealing the nicks afterward with phage T4 DNA ligase (NEB) in presence of 250 μg/ml of chloroquine.

Techniques: Electrophoresis, Western Blot, Two-Dimensional Gel Electrophoresis, Agarose Gel Electrophoresis

Journal: Chemical Science

Article Title: Target-fueled DNA walker for highly selective miRNA detection DNA walker for highly selective miRNA detection †Electronic supplementary information (ESI) available: DNA strand structure and sequences, assembly of DNA strands as noted in the text. See DOI: 10.1039/c5sc02784eClick here for additional data file.

doi: 10.1039/c5sc02784e

Figure Lengend Snippet: (A) Optimization of T4 DNA ligase concentration and time. (B) Optimization of phi29 DNA polymerase concentration and time. (C) Optimization of Nb.Mva1269I concentration and time. The assays were carried out in the reaction buffer, containing 10 nM let-7a, and 200 nM MB.

Article Snippet: Reagents and materials T4 DNA ligase and phi29 polymerase (10 units per μL) were obtained from New England Biolabs (Beijing, China).

Techniques: Concentration Assay

Journal: Chemical Science

Article Title: Target-fueled DNA walker for highly selective miRNA detection DNA walker for highly selective miRNA detection †Electronic supplementary information (ESI) available: DNA strand structure and sequences, assembly of DNA strands as noted in the text. See DOI: 10.1039/c5sc02784eClick here for additional data file.

doi: 10.1039/c5sc02784e

Figure Lengend Snippet: Schematic representation of the miRNA-responsive DNA walking nanostructure based on strand displacement cascades. (1) miRNA bound to branch A through toehold-mediated strand displacement, pushing the formed helices between the arm of strand C1 and the track to separate. (2) The released arm of strand C1 walked over to branch B, and the arm of strand C2 from branch B to branch C. (3) C1 and C2 were circulated using T4 DNA ligase. (4) The RCA reactions were performed. (5) The RCA product and the MB hybridized to each other, forming double-stranded DNA. The nicking enzyme catalyzed the multiple cleavage of the molecular beacon, resulting in the generation of a fluorescence signal.

Article Snippet: Reagents and materials T4 DNA ligase and phi29 polymerase (10 units per μL) were obtained from New England Biolabs (Beijing, China).

Techniques: Fluorescence

Journal: Biomicrofluidics

Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement

doi: 10.1063/1.4882775

Figure Lengend Snippet: AFM images of DNA-DNA crossings. (a) Bare DNA (3.8 kbp). (b) and (c) DNA with T4 DNA ligase andATP. Solid arrows indicate higher crossings consistent with ligase binding, outlined arrows indicateshallower crossings consistent with bare DNA.

Article Snippet: The majority of recent publications in the field use T4 DNA ligase supplied by New England Biolabs,which contains BSA.

Techniques: Binding Assay

Journal: Biomicrofluidics

Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement

doi: 10.1063/1.4882775

Figure Lengend Snippet: Mean aligned DNA molecule loop lengths as function of time for 22 molecules per dataset withtheir linear fits. Bare λ-DNA (blue), λ-DNA with T4 DNA ligase (green), and λ-DNA with T4 DNA ligaseand ATP (red).

Article Snippet: The majority of recent publications in the field use T4 DNA ligase supplied by New England Biolabs,which contains BSA.

Techniques:

Journal: Biomicrofluidics

Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement

doi: 10.1063/1.4882775

Figure Lengend Snippet: Histogram of end-to-end lengths of extended DNA molecules, bare λ-DNA (solid bars), λ-DNA with T4DNA ligase (gray bars), and λ-DNA with T4 DNA ligase and ATP (white bars). A Gaussian was fit toeach distribution to determine the

Article Snippet: The majority of recent publications in the field use T4 DNA ligase supplied by New England Biolabs,which contains BSA.

Techniques:

Journal: Biomicrofluidics

Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement

doi: 10.1063/1.4882775

Figure Lengend Snippet: Histograms of heights of DNA-DNA crossings. (a) Bare DNA (N = 41). (b) DNA with T4 DNA ligase andATP (N = 174). The red dotted line corresponds to unoccupied crossings, the blue dashed line tooccupied crossings, and the

Article Snippet: The majority of recent publications in the field use T4 DNA ligase supplied by New England Biolabs,which contains BSA.

Techniques:

Journal: Journal of Nucleic Acids

Article Title: Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining

doi: 10.4061/2010/823917

Figure Lengend Snippet: DNA end joining with immunodepleted extracts. HeLa WCE was immunodepleted for the various potential NHEJ proteins indicated, and immunodepletion (≥ 3-fold) of the target protein was confirmed by western blot (data not shown). The individual immunodepleted extracts were then assayed for DNA end-joining activity at 30°C for 2 hrs. (a) Results of DNA end-joining reactions performed in the absence of 5% PEG. (b) Results of DNA end-joining reactions performed in the presence of 5% PEG. All reactions were performed in triplicate and error bars indicate the standard deviation. (c) Wortmannin-insensitive DNA end joining is detectable in the DNA-PK cs immunodepleted HeLa WCE in the absence of PEG. Reactions were run in the absence of 5% PEG and where indicated, in the presence of 10 μ M wortmannin at 30°C for 2 hrs. (L) T4 DNA ligase positive control; (−) negative control; (ID) DNA-PK cs -immunodepleted WCE.

Article Snippet: Materials T4 DNA ligase (10 U/μ L) was purchased from Invitrogen (Carlsbad, CA.).

Techniques: Non-Homologous End Joining, Western Blot, Activity Assay, Standard Deviation, Positive Control, Negative Control

Journal: Nucleic Acids Research

Article Title: Real-time monitoring of nucleic acid ligation in homogenous solutions using molecular beacons

doi: 10.1093/nar/gng146

Figure Lengend Snippet: Ligation assay by T4 DNA ligase. Time scan curves of liga tion catalyzed by various concentrations of T4 DNA ligase. The curves from top to bottom are those obtained with different T4 DNA ligase concentrations: 2.3, 0.23, 0.115, 6.9 × 10 –2 , 2.3 × 10 –2 , 1.2 × 10 –2 , 6.9 × 10 –3 , 2.3 × 10 –3 , 1.2 × 10 –3 and 2.3 × 10 –4 U/ml. (Insert) The initial ligation velocity is plotted as a function of the concentration of T4 DNA ligase.

Article Snippet: Two samples containing 10 µl reaction solutions were moved from each sample before adding T4 DNA ligase, and after the ligation process had taken place for 360 s with the addition of ligase.

Techniques: Ligation, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Real-time monitoring of nucleic acid ligation in homogenous solutions using molecular beacons

doi: 10.1093/nar/gng146

Figure Lengend Snippet: Real-time fluorescence scans and corresponding gel electrophoresis. (Left) Curve A is a time scan of fluorescence intensity of MB with N1; B is of MB with N2 and N4; C is of MB with N2 and N3; curve D is of MB itself. t 0 is the time when T4 DNA ligase is added into the MB/oligo solution. (Right) Gel electrophoresis images. Lanes 1 and 2 are for sample D; 3 and 4 for sample C; 5 and 6 for sample B; and 7 and 8 for sample A. Lanes 1, 3, 5 and 7 represent samples D, C, B and A before the addition of T4 DNA ligase, while lanes 2, 4, 6 and 8 represent corresponding samples obtained at 360 s after the addition of ligase. There is a major difference between lanes 5 and 6, while there is basically no difference for all the other pairs.

Article Snippet: Two samples containing 10 µl reaction solutions were moved from each sample before adding T4 DNA ligase, and after the ligation process had taken place for 360 s with the addition of ligase.

Techniques: Fluorescence, Nucleic Acid Electrophoresis

Journal: Biochemistry and Biophysics Reports

Article Title: Efficient DNA ligation by selective heating of DNA ligase with a radio frequency alternating magnetic field

doi: 10.1016/j.bbrep.2016.10.006

Figure Lengend Snippet: Dependence of the efficiency of DNA ligation using T4 DNA ligase immobilized on ferromagnetic particles in the absence of a magnetic field on the ambient temperature. The ordinate axis represents the ligation efficiency, which is normalized by that at 16 °C. The standard deviations are obtained from 6 independent experiments.

Article Snippet: Five μL of T4 DNA ligase/ferromagnetic particle hybrid-dispersed solution, 2 μL of T4 DNA ligase buffer (Takara Bio Inc.), which consisted of 660 mM Tris-HCl (pH 7.6), 66 mM MgCl2 , 100 mM DTT and 1 mM ATP, 5 μL of aqueous solution containing 0.4 mM each of the DNA fragments, and 8 μL of sterilized water were mixed in a test tube, which was placed in a cylindrical container filled with circulating water, the temperature of which was regulated at 16 °C, from a constant-temperature bath (LTB-400, AS ONE CO.).

Techniques: DNA Ligation, Ligation

Journal: Biochemistry and Biophysics Reports

Article Title: Efficient DNA ligation by selective heating of DNA ligase with a radio frequency alternating magnetic field

doi: 10.1016/j.bbrep.2016.10.006

Figure Lengend Snippet: Dependence of the efficiency of DNA ligation using T4 DNA ligase immobilized on ferromagnetic particles under an ac magnetic field of 0.34 MHz on the amplitude of the magnetic field. The ambient temperature is 16 °C. The ordinate axis represents the ligation efficiency under an ac magnetic field, which is normalized by that in the absence of a magnetic field. The inset shows the ligation efficiency under the ac magnetic field as a function of the average surface temperature of ferromagnetic particles, noting that the surface temperature increases with an increase in the field amplitude. The standard deviations are obtained from 6 independent experiments.

Article Snippet: Five μL of T4 DNA ligase/ferromagnetic particle hybrid-dispersed solution, 2 μL of T4 DNA ligase buffer (Takara Bio Inc.), which consisted of 660 mM Tris-HCl (pH 7.6), 66 mM MgCl2 , 100 mM DTT and 1 mM ATP, 5 μL of aqueous solution containing 0.4 mM each of the DNA fragments, and 8 μL of sterilized water were mixed in a test tube, which was placed in a cylindrical container filled with circulating water, the temperature of which was regulated at 16 °C, from a constant-temperature bath (LTB-400, AS ONE CO.).

Techniques: DNA Ligation, Ligation

Journal: PLoS ONE

Article Title: High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

doi: 10.1371/journal.pone.0156106

Figure Lengend Snippet: pAK-TAG expression vector and high level expression of recombinant AK fusion proteins in soluble form. (A) Schematic representation of the pAK-TAG vector. (B) SDS-PAGE analysis of the expression of AK-TNFα, AK-TRAIL, and AK-T4 DNA ligase.

Article Snippet: Chemicals T4 DNA ligase, Taq polymerase, and restriction enzymes were obtained from Takara.

Techniques: Expressing, Plasmid Preparation, Recombinant, SDS Page

Journal: Nucleic Acids Research

Article Title: HMGB1 interacts with human topoisomerase II? and stimulates its catalytic activity

doi: 10.1093/nar/gkm525

Figure Lengend Snippet: HMGB1 promotes intermolecular association of DNA. ( A ) Macromolecular crowding favors intermolecular ligase-mediated DNA end-joining by HMGB1. Linearized plasmid pTZ19R (∼15 nM) was pre-incubated with 0.5 μM (lanes 3 and 6) or 1.5 μM (lanes 4 and 7) HMGB1, and then treated with 0.2 U of T4 DNA ligase in the presence (lanes 6 and 7) or absence (lanes 3 and 4) of 5% polyethyleneglycol (PEG). L2, dimers; L3 trimers or higher multimers. Linear, linearized plasmid pBR322; circular, closed-circular plasmid pBR322. ( B ) HMGB1 promotes topo IIα-catalyzed interlocking of DNA into multimers (catenanes) in the presence of PEG. Supercoiled plasmid pTZ19R (∼15 nM, lane 1) was pre-incubated with HMGB1 (4.5 μM) in the absence or presence of PEG (as indicated), and treated with topo IIα (∼7 nM). ( C ) Both relaxed and supercoiled plasmid DNAs form multimers with HMGB1 and topo IIα. Relaxed or supercoiled plasmids pTZ19R (∼15 nM) were pre-incubated with 0.5 μM (lanes 3 and 7), 1.5 μM (lanes 4 and 8) and 4.5 μM HMGB1 (lanes 5 and 9) in the presence of 5% PEG, followed by treatment with topo IIα (∼7 nM). ( D ) DNA multimers formed by topo IIα and HMGB1 are catenanes. Reactions from (C) (lane 4) were deproteinized and treated with increasing amounts of topo IIα (10 and 20 nM, left to right) for 30 min at 37°C. Deproteinized samples in (A–D) were separated on 1% agarose gels, and the resolved DNA samples were visualized by ethidium bromide staining as detailed in Materials and Methods section. The gels are presented as negatives. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA; FIII, linearized plasmid DNA ( Hin dIII).

Article Snippet: The DNA was then ligated with 0.2 U of T4 DNA ligase in a final volume of 20 μl at 30°C for 30 min in the presence or absence of 5% (w/v) polyethylene glycol (PEG 8000, Sigma).

Techniques: Plasmid Preparation, Incubation, Staining

Journal: Nucleic Acids Research

Article Title: Identification of recognition residues for ligation-based detection and quantitation of pseudouridine and N6-methyladenosine

doi: 10.1093/nar/gkm657

Figure Lengend Snippet: ( A ) Chemical structures of Ψ and m 6 A. ( B ) Scheme for T4 DNA ligase-catalyzed joining of two DNA substrates. In the ternary RNA/DNA complex, the black line corresponds to the 30-mer RNA template with the modified nucleotide (open circle) located at the 15th position. Blue lines correspond to the ligation substrates with the recognition residue shown as a filled blue circle.

Article Snippet: The optimized ligation reactions were carried out with 0.15 µM 30-mer RNA with or without Ψ or m6 A modifications, 0.5 µM floater and 0.38 µM of 5′-32 P-labeled anchor in 66 mM Tris–HCl, pH 7.6, 0.5 mM ZnCl2 , 10 mM DTT, 66 µM ATP, 15% DMSO and 0.25 U/µl T4 DNA ligase (USB Inc.).

Techniques: Modification, Ligation

Journal: Scientific Reports

Article Title: Transcription activator-like effector nuclease-mediated transduction of exogenous gene into IL2RG locus

doi: 10.1038/srep05043

Figure Lengend Snippet: TALEN-induced genomic mutation in IL2RG . (a) T7 endonuclease I assay using TALENs for Jurkat cells. Jurkat cells were transfected with TALEN expression vectors by electroporation. After 5 days culture, genomic DNA was isolated and the TALEN target locus was amplified by PCR. A T7 endonuclease I assay was performed using purified PCR products. The arrowhead indicates the expected position of the digested products in the agarose gel. (b) Sequencing results of the PCR fragments, revealing different mutations in the TALEN target site. Jurkat cells were cultured for 5 days after electroporation, and cloning was performed by limiting dilution. Genomic DNA was isolated from cloned Jurkat cells and DNA sequencing was performed. Sequences for wild-type (WT) and deletion mutants (del1–4) are shown. (c) Functional analysis of genome-modified Jurkat cells. The level of IL2RG expression in genome-modified Jurkat cells was analyzed using flow cytometry. Cells were incubated with APC-conjugated-anti-hCD132 antibody for IL2RG and APC-IgG2b antibody as an isotype control. MFI, Mean Fluorescence Intensity of CD132. (d) qPCR analysis of BCL2 . BCL2 expression was examined 48 hr after the PMA and ionomycin stimulation in the presence of exogenous IL-2. Data are shown as mean ± SD (n = 3).

Article Snippet: The pVenus vector was used as the backbone to construct the IL2RG -targeting vector pVenus-L. A 5662 bp 5′ homology arm and a 3000 bp 3′ homology arm were amplified by PCR from the Jurkat cell genome, and cloned into the pVenus by using a DNA Ligation Kit.

Techniques: Mutagenesis, T7EI Assay, TALENs, Transfection, Expressing, Electroporation, Isolation, Amplification, Polymerase Chain Reaction, Purification, Agarose Gel Electrophoresis, Sequencing, Cell Culture, Clone Assay, DNA Sequencing, Functional Assay, Modification, Flow Cytometry, Cytometry, Incubation, Fluorescence, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Transcription activator-like effector nuclease-mediated transduction of exogenous gene into IL2RG locus

doi: 10.1038/srep05043

Figure Lengend Snippet: TALEN-mediated genome editing. (a) Top : Schematic of the endogenous IL2RG locus. Hind III, Hind III restriction sites used for Southern blot analysis; TM, transmembrane domain. Middle : Schematic of the targeting vector. The targeting vector contained Venus cDNA, lox-P-flanked Neomycin resistance cassette, and DT-A negative selectable marker. The 5′ homology arm upstream of the IL2RG start codon was cloned upstream of Venus cDNA, and the 3′ homology arm downstream of the IL2RG transmembrane sequence (exon 6) was cloned downstream of the Neomycin resistance cassette (Neor). 5′ probe, Probe used for Southern blot analysis. Bottom : Schematic of the targeted IL2RG locus. A novel Hind III restriction site would introduced when the targeted knock-in was successful. (b) Flow cytometric analysis of Venus in Jurkat cells with targeted knock-in of IL2RG . Each Jurkat cell clone was analyzed for YFP fluorescence expressed from knocked-in Venus cDNA. WT, wild-type Jurkat cells; KI 1-4, individual clones with targeted knock-in; KI 5, Venus hi ; MFI, Mean Fluorescence Intensity of Venus. (c) Southern blot analysis of Jurkat cells with targeted knock-in of IL2RG . Hind III digestion resulted in a 3788 bp band from the WT endogenous IL2RG locus and a 3515 bp band (containing Venus cDNA and the Neomycin resistance cassette) from the targeted knock-in. Targeting vector was used as positive control for targeted knock-in, and genomic DNA isolated from WT Jurkat cells was used as negative control.

Article Snippet: The pVenus vector was used as the backbone to construct the IL2RG -targeting vector pVenus-L. A 5662 bp 5′ homology arm and a 3000 bp 3′ homology arm were amplified by PCR from the Jurkat cell genome, and cloned into the pVenus by using a DNA Ligation Kit.

Techniques: Southern Blot, Plasmid Preparation, Marker, Clone Assay, Sequencing, Knock-In, Flow Cytometry, Fluorescence, Positive Control, Isolation, Negative Control