t4 dna ligase Search Results


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  • 99
    New England Biolabs t4 dna ligase
    Ligation experiment of gapped pGLuc1temGG (left) and pGLuc2temGTG (right) in presence of different insertion strands with <t>T4</t> DNA Ligase at 16°C for 12 h; lane 1: gapped pGLuc1temGG alone, lane 2: plasmid + 13-is, lane 3: plasmid + 13-is-Pt, lane
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 50053 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher t4 dna ligase
    The kinetics of the ligation reaction in the presence or absence of SDS. Electrophoretic separation of the products obtained upon the ligation of pUC18 Hind III fragments for the indicated times in the presence or absence of SDS and Triton X-100 in an ethidium bromide-stained agarose gel. The reaction was carried out in 1× <t>T4</t> DNA Ligase Buffer (Fermentas) with 100 ng/ µl DNA and 0.1 U/ µl T4 DNA ligase (Fermentas). M–DNA size marker (Fermentas, SM0331).
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25057 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega t4 dna ligase
    Ligation in the presence of DNA-PK requires ATP hydrolysis and an active DNA-PK CS kinase. ( A ) An overall labeled DNA substrate with cohesive ends was incubated with <t>T4</t> DNA ligase, either in the absence (lanes 1–3) or the presence (lanes 4 and 5) of DNA-PK. ATP or AMP-PNP was present as indicated. Ligation products were separated by agarose gel electrophoresis. The nature of the ligation products, identified as intra- or inter-molecular ligation products, was confirmed by exonuclease V digestion. Note that intra-molecular ligation products can be either ligated on one strand (open circular form) or on both strands (covalently closed circular form). ( B ) An overall labeled DNA substrate with cohesive ends was incubated with E.coli DNA ligase, either in the absence (lanes 1–4) or the presence (lanes 5 and 6) of DNA-PK. ATP and/or NAD + were present as indicated. ( C ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of DNA-PK. All reaction mixtures contained ATP. The DNA-PK CS kinase inhibitor wortmannin was added in lane 4. Total levels of ligation products in all lanes were decreased in comparison with (A), due to the presence of DMSO in the reaction mixtures. ( D ) Wortmannin inhibits autophosphorylation of DNA-PK CS . Incorporation of radiolabeled phosphate into DNA-PK CS was determined in the absence and presence of 1 or 10 µM wortmannin. Even 1 µM wortmannin completely inhibits DNA-PK CS autophosphorylation.
    T4 Dna Ligase, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 11518 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa t4 dna ligase
    Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by <t>T4</t> DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.
    T4 Dna Ligase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 7380 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 ligase
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
    T4 Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5002 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa dna ligation kit
    TALEN-induced genomic mutation in IL2RG . (a) T7 endonuclease I assay using TALENs for <t>Jurkat</t> cells. Jurkat cells were transfected with TALEN expression vectors by electroporation. After 5 days culture, genomic <t>DNA</t> 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).
    Dna Ligation Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 2463 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs taq dna ligase
    FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified <t>NC-DNA</t> (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and <t>Taq</t> DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P
    Taq Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1648 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dna ligase
    Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease <t>III</t> in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid <t>DNA</t> which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.
    Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1336 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 dna ligase reaction buffer
    Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, <t>T4</t> ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).
    T4 Dna Ligase Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1354 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim t4 dna ligase
    T4 DNA-ligase activity in the presence of SA. T4 DNA-ligase was treated or not with increasing SA concentrations (15 min at 25°C) and then incubated at 37°C for 1 min with the oligo substrate. ( A ) The oligo(dT) 16  multimers were separated in polyacrylamide/urea gels: T4 DNA-ligase without SA (lane 1) or incubated with increasing SA concentrations of 2.5(2), 5(3), 10(4), and 20(5) μM. ( B ) The activity was quantitated using an InstantImager (Packard). ( C ) Inhibition of enzyme-adenylate formation by SA. T4 DNA-ligase was incubated or not with increasing SA concentrations (15 min at 25°C) before the addition of [α- 32 P]ATP. The enzyme adenylate complexes were separated by electrophoresis and detected by autoradiography.
    T4 Dna Ligase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 908 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rapid dna ligation kit
    SCIPay design permits CRISPR-guided integration of specific donor <t>DNA</t> sequences via homology directed repair. (A) Overview of SCIPay design. Here, HA present in the payload sequence lead to its genomic integration via HDR. (B – G) EL4 cells were electroporated with SCIPay containing a <t>BFP</t> transgene targeting B2M with various HA lengths. (B) Percentage of knock-out cells. (C) Efficiency and (D) precision scores for GFP, denoting integration of the non-payload plasmid sequence. (E) Efficiency and (F) precision scores for BFP, denoting HDR integration of BFP. (G) Ratio of BFP-to GFP-positive cells. (H) Representative flow cytometry plots. Results in (B – G) represent means +/- SEM of 4 independent experiments. Data was analyzed using 1-way ANOVA and significant differences are indicated with lower case letters, where groups with different letters are significantly different than each other (p
    Rapid Dna Ligation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1352 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzymatics t4 dna ligase
    3′ Branch ligation by <t>T4</t> DNA ligase at non-conventional DNA ends formed by nicks, gaps, and overhangs. (a) Schematic representation of ligation assay with different DNA accepter types. The blunt-end DNA donor (blue) is a synthetic, partially dsDNA molecule with dideoxy 3 ′ -termini (filled circles) to prevent DNA donor self-ligation. The long arm of the donor is 5 ′- phosporylated. The DNA acceptors were assembled using 2 or 3 oligos (black, red, and orange lines) to form a nick (without phosphates), a gap (1 or 8 nt), or a 36-nt 3 ′ -recessive end. All strands of the substrates are unphosphorylated, and the scaffold strand is 3 ′ dideoxy protected. (b) Analysis of the size shift of ligated products of substrates 1, 2, 3, and 4, respectively, using a 6% denaturing polyacrylamide gel. Reactions were performed according to the optimized condition. The negative no-ligase controls (lanes 1, 3, 4, 6, 7, 9, 10, 12, and 13) were loaded at 1 or 0.5× volume of corresponding experimental assays. If ligation occurs, the substrate size is shifted up by 22 nt. Red arrowheads correspond to the substrate, and purple arrowheads correspond to donor-ligated substrates. Donor and substrate sequences in Supplementary Table S1 . (c) Expected sizes of substrate and ligation product and approximate ligation efficiency in each experimental group. The intensity of each band was estimated using ImageJ and normalized by its expected size. Ligation efficiency was estimated by dividing the normalized intensity of ligated products by the normalized total intensity of ligated and unligated products.
    T4 Dna Ligase, supplied by Enzymatics, used in various techniques. Bioz Stars score: 93/100, based on 863 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher t4 dna ligase buffer
    Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard <t>T4</t> DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.
    T4 Dna Ligase Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 847 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dna ligase
    <t>RNAse</t> protection assay. The upper panel shows a schematic representation of part of the 5′ region of the human CD45 gene and the RNA probes generated in vitro . The lower panel shows the protected fragments in different cell lines: Jurkat (human T cell), Raji (human B cell), K562 (human erythroid), U937 (human myeloid), EL-4 (mouse T cell) and HFB-1 (human B cell). M is the Hin fI-digested φX174 <t>DNA</t> used as a marker. The size of the bands in bp is indicated at both sides.
    Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1000 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore t4 dna ligase
    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 <t>T4</t> 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).
    T4 Dna Ligase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 625 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t7 dna ligase
    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 <t>T4</t> 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).
    T7 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 404 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare t4 dna ligase
    Effect of DNA ligase addition on the DNA ligase-[α- 32 P]AMP reduction. ( A ) Cell-free DNA repair assay was carried out for 60 min with HeLa S3 cell extracts containing DNA ligase-[α- 32 P]AMP. Plasmid DNA containing either γ-ray-induced SSIs (γ-SSI) or alkylated base damage induced by MNNG was used for the assay. The reactions also contained Lig I, Lig III or <t>T4</t> DNA ligase (T4 Lig). After the repair reaction, the mixtures were fractionated on an SDS–7.5% polyacrylamide gel, and 32 P activity was visualized by autoradiography. Alternatively, 32 P activities were measured with an AlphaImager (Packard) and quantified results are shown in ( B ) (γ-SSI) and ( C ) (MNNG). The results shown are from one of four independent experiments. The amount of Lig I-[ 32 P]AMP (non-damaged DNA) was calculated as 100%.
    T4 Dna Ligase, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 530 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo t4 dna ligase
    Effect of DNA ligase addition on the DNA ligase-[α- 32 P]AMP reduction. ( A ) Cell-free DNA repair assay was carried out for 60 min with HeLa S3 cell extracts containing DNA ligase-[α- 32 P]AMP. Plasmid DNA containing either γ-ray-induced SSIs (γ-SSI) or alkylated base damage induced by MNNG was used for the assay. The reactions also contained Lig I, Lig III or <t>T4</t> DNA ligase (T4 Lig). After the repair reaction, the mixtures were fractionated on an SDS–7.5% polyacrylamide gel, and 32 P activity was visualized by autoradiography. Alternatively, 32 P activities were measured with an AlphaImager (Packard) and quantified results are shown in ( B ) (γ-SSI) and ( C ) (MNNG). The results shown are from one of four independent experiments. The amount of Lig I-[ 32 P]AMP (non-damaged DNA) was calculated as 100%.
    T4 Dna Ligase, supplied by Toyobo, used in various techniques. Bioz Stars score: 92/100, based on 339 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Ligation experiment of gapped pGLuc1temGG (left) and pGLuc2temGTG (right) in presence of different insertion strands with T4 DNA Ligase at 16°C for 12 h; lane 1: gapped pGLuc1temGG alone, lane 2: plasmid + 13-is, lane 3: plasmid + 13-is-Pt, lane

    Journal: Bioconjugate chemistry

    Article Title: Preparation of Mammalian Expression Vectors Incorporating Site-Specifically Platinated-DNA Lesions

    doi: 10.1021/bc900031a

    Figure Lengend Snippet: Ligation experiment of gapped pGLuc1temGG (left) and pGLuc2temGTG (right) in presence of different insertion strands with T4 DNA Ligase at 16°C for 12 h; lane 1: gapped pGLuc1temGG alone, lane 2: plasmid + 13-is, lane 3: plasmid + 13-is-Pt, lane

    Article Snippet: The reaction was supplemented with T4 DNA ligase (80 U) in buffer (50 mM Tris·HCl pH 7.6, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP) and incubated at 16 °C for 12 h. The mixture was supplemented with 6X loading buffer (NEB), heated at 75 °C for 15 min and separated using preparative 0.8% w/v agarose gel electrophoresis containing 0.5 µg/mL EtdBr.

    Techniques: Ligation, Plasmid Preparation

    Schematic overview of the QL cloning procedure. An envelope gene or an envelope library is amplified with primers to introduce flanking Esp3I restriction sites enabling the generation of a 5′ NcoI and a 3′ Xho sitey (A; top). The envelope gene or an envelope library is incubated together with pQL9/11 in a one-tube reaction with Esp3I and T4-Ligase. Compatible “sticky-ends” (equally colored) can be ligated successfully, direct proper orientation and mediating resistance for further cleavage (A). Following transformation of CcdB sensitive bacteria, only recipients bearing a plasmid without CcdB are able to form colonies in the presence of ampicillin. (B) The lentiviral vector construct pQL9 comprises (i) 5′LTR (Long terminal repeat), (ii) EF1α (human promotor), (iii) GFP (marker gene), (iv) an IRES (internal ribosome entry site), (v) a CcdB positive selection marker [58] , and (vi) a 3′LTR sequence.

    Journal: PLoS ONE

    Article Title: A Mammalian Cell Based FACS-Panning Platform for the Selection of HIV-1 Envelopes for Vaccine Development

    doi: 10.1371/journal.pone.0109196

    Figure Lengend Snippet: Schematic overview of the QL cloning procedure. An envelope gene or an envelope library is amplified with primers to introduce flanking Esp3I restriction sites enabling the generation of a 5′ NcoI and a 3′ Xho sitey (A; top). The envelope gene or an envelope library is incubated together with pQL9/11 in a one-tube reaction with Esp3I and T4-Ligase. Compatible “sticky-ends” (equally colored) can be ligated successfully, direct proper orientation and mediating resistance for further cleavage (A). Following transformation of CcdB sensitive bacteria, only recipients bearing a plasmid without CcdB are able to form colonies in the presence of ampicillin. (B) The lentiviral vector construct pQL9 comprises (i) 5′LTR (Long terminal repeat), (ii) EF1α (human promotor), (iii) GFP (marker gene), (iv) an IRES (internal ribosome entry site), (v) a CcdB positive selection marker [58] , and (vi) a 3′LTR sequence.

    Article Snippet: Meanwhile a second reaction for the ligation was prepared. (II) 3 µL 10 mM ATP, 1 µL 10 x Tango Buffer, 1 µL 10 mM DTT, 1 µL T4-Ligase (NEB) addition of H2 0 to reach 10 µL.

    Techniques: Clone Assay, Amplification, Introduce, Incubation, Transformation Assay, Plasmid Preparation, Construct, Marker, Selection, Sequencing

    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

    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

    The kinetics of the ligation reaction in the presence or absence of SDS. Electrophoretic separation of the products obtained upon the ligation of pUC18 Hind III fragments for the indicated times in the presence or absence of SDS and Triton X-100 in an ethidium bromide-stained agarose gel. The reaction was carried out in 1× T4 DNA Ligase Buffer (Fermentas) with 100 ng/ µl DNA and 0.1 U/ µl T4 DNA ligase (Fermentas). M–DNA size marker (Fermentas, SM0331).

    Journal: PLoS ONE

    Article Title: Actual Ligation Frequencies in the Chromosome Conformation Capture Procedure

    doi: 10.1371/journal.pone.0060403

    Figure Lengend Snippet: The kinetics of the ligation reaction in the presence or absence of SDS. Electrophoretic separation of the products obtained upon the ligation of pUC18 Hind III fragments for the indicated times in the presence or absence of SDS and Triton X-100 in an ethidium bromide-stained agarose gel. The reaction was carried out in 1× T4 DNA Ligase Buffer (Fermentas) with 100 ng/ µl DNA and 0.1 U/ µl T4 DNA ligase (Fermentas). M–DNA size marker (Fermentas, SM0331).

    Article Snippet: Next, 100 U of T4 DNA Ligase (Fermentas) was added, and the DNA was ligated for 4.5 h at 16°C and then for 30 min at room temperature with slow agitation.

    Techniques: Ligation, Staining, Agarose Gel Electrophoresis, Marker

    Ligation in the presence of DNA-PK requires ATP hydrolysis and an active DNA-PK CS kinase. ( A ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1–3) or the presence (lanes 4 and 5) of DNA-PK. ATP or AMP-PNP was present as indicated. Ligation products were separated by agarose gel electrophoresis. The nature of the ligation products, identified as intra- or inter-molecular ligation products, was confirmed by exonuclease V digestion. Note that intra-molecular ligation products can be either ligated on one strand (open circular form) or on both strands (covalently closed circular form). ( B ) An overall labeled DNA substrate with cohesive ends was incubated with E.coli DNA ligase, either in the absence (lanes 1–4) or the presence (lanes 5 and 6) of DNA-PK. ATP and/or NAD + were present as indicated. ( C ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of DNA-PK. All reaction mixtures contained ATP. The DNA-PK CS kinase inhibitor wortmannin was added in lane 4. Total levels of ligation products in all lanes were decreased in comparison with (A), due to the presence of DMSO in the reaction mixtures. ( D ) Wortmannin inhibits autophosphorylation of DNA-PK CS . Incorporation of radiolabeled phosphate into DNA-PK CS was determined in the absence and presence of 1 or 10 µM wortmannin. Even 1 µM wortmannin completely inhibits DNA-PK CS autophosphorylation.

    Journal: Nucleic Acids Research

    Article Title: The role of DNA dependent protein kinase in synapsis of DNA ends

    doi: 10.1093/nar/gkg889

    Figure Lengend Snippet: Ligation in the presence of DNA-PK requires ATP hydrolysis and an active DNA-PK CS kinase. ( A ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1–3) or the presence (lanes 4 and 5) of DNA-PK. ATP or AMP-PNP was present as indicated. Ligation products were separated by agarose gel electrophoresis. The nature of the ligation products, identified as intra- or inter-molecular ligation products, was confirmed by exonuclease V digestion. Note that intra-molecular ligation products can be either ligated on one strand (open circular form) or on both strands (covalently closed circular form). ( B ) An overall labeled DNA substrate with cohesive ends was incubated with E.coli DNA ligase, either in the absence (lanes 1–4) or the presence (lanes 5 and 6) of DNA-PK. ATP and/or NAD + were present as indicated. ( C ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of DNA-PK. All reaction mixtures contained ATP. The DNA-PK CS kinase inhibitor wortmannin was added in lane 4. Total levels of ligation products in all lanes were decreased in comparison with (A), due to the presence of DMSO in the reaction mixtures. ( D ) Wortmannin inhibits autophosphorylation of DNA-PK CS . Incorporation of radiolabeled phosphate into DNA-PK CS was determined in the absence and presence of 1 or 10 µM wortmannin. Even 1 µM wortmannin completely inhibits DNA-PK CS autophosphorylation.

    Article Snippet: First, we used AMP-PNP, an ATP analog that supports activity of T4 DNA ligase, but cannot function as a cofactor for DNA-PKCS (Fig. A).

    Techniques: Ligation, Labeling, Incubation, Agarose Gel Electrophoresis

    Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.

    Journal: PLoS ONE

    Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

    doi: 10.1371/journal.pone.0138774

    Figure Lengend Snippet: Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.

    Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

    Techniques: DNA Ligation, Labeling, Incubation, Ligation, Electrophoresis

    Histone H1 inhibits the ability of HMGB1 to bend DNA. A , formation of DNA circles by HMGB1 is inhibited by the full-length histone H1 (DNA circularization assay). The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 5 nM HMGB1, followed by titration with increasing concentrations of H1 (0.2–15 nM, left to right ) and ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. Panels B - E , DNA circularization assays in the presence of the full-length histone H1(fl) or peptides H1Δ24, H1Δ48 and H1Δ72. The percentage of DNA circles by reduced or oxidized HMGB1 or HMGB1ΔC (50 nM) in the presence of increasing concentrations of H1 or H1 peptides (1–15 nM, left to right ) is indicated. The percentage of the minicircles formed by HMGB1 or HMGB1ΔC in the absence of H1 or peptides was arbitrary set to 100%. Oxidized HMGB1 or HMGB1ΔC proteins are indicated in red.

    Journal: PLoS ONE

    Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

    doi: 10.1371/journal.pone.0138774

    Figure Lengend Snippet: Histone H1 inhibits the ability of HMGB1 to bend DNA. A , formation of DNA circles by HMGB1 is inhibited by the full-length histone H1 (DNA circularization assay). The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 5 nM HMGB1, followed by titration with increasing concentrations of H1 (0.2–15 nM, left to right ) and ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. Panels B - E , DNA circularization assays in the presence of the full-length histone H1(fl) or peptides H1Δ24, H1Δ48 and H1Δ72. The percentage of DNA circles by reduced or oxidized HMGB1 or HMGB1ΔC (50 nM) in the presence of increasing concentrations of H1 or H1 peptides (1–15 nM, left to right ) is indicated. The percentage of the minicircles formed by HMGB1 or HMGB1ΔC in the absence of H1 or peptides was arbitrary set to 100%. Oxidized HMGB1 or HMGB1ΔC proteins are indicated in red.

    Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

    Techniques: Labeling, Incubation, Titration, Ligation, Electrophoresis

    The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1ΔC on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 2, 5, 10, 15, 25, 50 and 100 nM of HMGB1 lacking the acidic C-tail (HMGB1ΔC, left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced (black triangle) or oxidized (empty triangle) HMGB1ΔC, as compared to DNA circles formed under the same conditions by reduced (black circles) or oxidized (empty circles) full-length HMGB1. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1ΔC, oxidized HMGB1ΔC, and HMGB1ΔC(F37A). Concentrations of proteins were 5, 10, 25, 50 and 100 nM ( left to right ).

    Journal: PLoS ONE

    Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

    doi: 10.1371/journal.pone.0138774

    Figure Lengend Snippet: The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1ΔC on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 2, 5, 10, 15, 25, 50 and 100 nM of HMGB1 lacking the acidic C-tail (HMGB1ΔC, left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced (black triangle) or oxidized (empty triangle) HMGB1ΔC, as compared to DNA circles formed under the same conditions by reduced (black circles) or oxidized (empty circles) full-length HMGB1. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1ΔC, oxidized HMGB1ΔC, and HMGB1ΔC(F37A). Concentrations of proteins were 5, 10, 25, 50 and 100 nM ( left to right ).

    Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

    Techniques: Mutagenesis, Labeling, Incubation, Ligation, Electrophoresis

    The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1 on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was preincubated with 2, 5, 10, 15, 25, 50 and 100 nM HMGB1 proteins ( left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced HMGB1, oxidized HMGB1 or HMGB1(Cys22A/Cys44A) mutant. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1 and HMGB1(F37A) mutant (5, 20, 50 and 100 nM HMGB1, left to right ). C22/C44, HMGB1(Cys22A/Cys44A) mutant.

    Journal: PLoS ONE

    Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

    doi: 10.1371/journal.pone.0138774

    Figure Lengend Snippet: The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1 on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was preincubated with 2, 5, 10, 15, 25, 50 and 100 nM HMGB1 proteins ( left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced HMGB1, oxidized HMGB1 or HMGB1(Cys22A/Cys44A) mutant. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1 and HMGB1(F37A) mutant (5, 20, 50 and 100 nM HMGB1, left to right ). C22/C44, HMGB1(Cys22A/Cys44A) mutant.

    Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

    Techniques: Mutagenesis, Labeling, Ligation, Electrophoresis

    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.

    Journal: PLoS ONE

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection

    doi: 10.1371/journal.pone.0037617

    Figure Lengend Snippet: Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.

    Article Snippet: Equal molar amounts (typically 250–500 ng at ∼ 100 – 250 ng/µl ) of orthogonally protected synthons were mixed, 0.5–1 unit T4 ligase (Fermentas) and T4 ligase buffer (Fermentas) were added and the ligation mixture was incubated for 10–20 min at 16°C.

    Techniques: Incubation, Plasmid Preparation, Clone Assay, Expressing, Construct

    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).

    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

    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.

    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

    FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Journal: PLoS Pathogens

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    doi: 10.1371/journal.ppat.1007124

    Figure Lengend Snippet: FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Article Snippet: NC-DNA (108 copies) was incubated with 32 units (U) of Thermostable FEN1 in ThermoPol Buffer (New England Biolabs) at 65°C for 10 min, followed by incubation with 8 U of Bst DNA polymerase, 40 U of Taq DNA ligase, 100 μM dNTPs, and NAD+ (all from New England Biolabs).

    Techniques: In Vitro, Tube Formation Assay, Purification, Incubation, Recombinant, Real-time Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing

    Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease III in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid DNA which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.

    Journal: The Journal of General Virology

    Article Title: Molecular differences between two Jeryl Lynn mumps virus vaccine component strains, JL5 and JL2

    doi: 10.1099/vir.0.013946-0

    Figure Lengend Snippet: Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease III in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid DNA which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.

    Article Snippet: Restriction enzymes, reverse transcriptase SuperScript III, high-fidelity Taq DNA polymerase, Pfu polymerase, Phusion DNA polymerase, Klenow fragment of DNA polymerase, exonuclease III and DNA ligase were obtained from New England Biolabs (NEB) or Invitrogen and used according to the manufacturers' instructions.

    Techniques: Ligation, Clone Assay, Plasmid Preparation, Sequencing, Mutagenesis, In Vitro, Methylation

    The large terminase gp17 is a weak endonuclease. ( A ) Time course of cleavage of circular pET28b plasmid (100 ng) by gp17 (1.5 µM). ( B ) Cleavage of topoisomerase 1 relaxed DNA by gp17. ( C ) Increasing concentrations of gp17 (9–900 nM) were incubated with circular pAd10 DNA (400 ng, 0.9 nM) and the amount of undigested circular DNA in each lane was quantified by laser densitometry. ( D ) Comparison of the nuclease activity of T4 gp17 with DNase I (non-specific nickase) and Sau3A1 (frequent cutting restriction endonuclease). Increasing concentrations of each enzyme were incubated with circular pAd10 DNA (400 ng, 0.9 nM) with the enzyme (monomer) to DNA ratio (number of molecules of each) varied over a range of 10–1000:1. The enzyme:DNA ratio at 50% cleavage was determined by quantifying the amount of undigested circular DNA in each lane. Values represent average of duplicates from two independent experiments. The ‘C’ lanes represent untreated DNA. See ‘Materials and Methods’ section for additional details.

    Journal: Nucleic Acids Research

    Article Title: Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4

    doi: 10.1093/nar/gkq1191

    Figure Lengend Snippet: The large terminase gp17 is a weak endonuclease. ( A ) Time course of cleavage of circular pET28b plasmid (100 ng) by gp17 (1.5 µM). ( B ) Cleavage of topoisomerase 1 relaxed DNA by gp17. ( C ) Increasing concentrations of gp17 (9–900 nM) were incubated with circular pAd10 DNA (400 ng, 0.9 nM) and the amount of undigested circular DNA in each lane was quantified by laser densitometry. ( D ) Comparison of the nuclease activity of T4 gp17 with DNase I (non-specific nickase) and Sau3A1 (frequent cutting restriction endonuclease). Increasing concentrations of each enzyme were incubated with circular pAd10 DNA (400 ng, 0.9 nM) with the enzyme (monomer) to DNA ratio (number of molecules of each) varied over a range of 10–1000:1. The enzyme:DNA ratio at 50% cleavage was determined by quantifying the amount of undigested circular DNA in each lane. Values represent average of duplicates from two independent experiments. The ‘C’ lanes represent untreated DNA. See ‘Materials and Methods’ section for additional details.

    Article Snippet: The digested DNA was precipitated by 8 M ammonium acetate and resuspended in water, followed by the addition of T4 DNA ligase (Fermentas) or E. coli DNA ligase (New England Biolabs) in a 25 µl reaction mixture.

    Techniques: Plasmid Preparation, Incubation, Activity Assay

    ATP stimulates gp17 nuclease. ( A ) Nuclease activity of gp17 is stimulated in presence of ATP. gp17 (1 µM) was incubated with linear pAd10 DNA (100 ng) in the presence of increasing concentrations of ATP (0.05–5 mM) . Note that the DNA is degraded to small fragments in some of the lanes. Therefore, very little DNA smear is seen in these lanes. ( B ) Nuclease activity of gp17 in the presence of ATP analogs. gp17 (1.2 µM) was incubated with circular pAd10 DNA (200 ng) in the presence of ATP, ADP, ATP-γS or AMP-PNP (1 mM). ( C ) The T4 nuclease domain (C360, amino acids 360–577) (left panel; lanes 1–7) or the RB49 nuclease domain (C360, amino acids 358–607) (right panel; lanes 8–14) is not stimulated by ATP. T4 C360 (4 µM) or RB49 C360 (1 µM), either alone (lanes 2 and 9) or in the presence of ATP (lanes 3–7 and 10–14) was incubated with linear pAd10 DNA (100 ng) for 15 min and the samples were analyzed by 0.8% (w/v) agarose gel electrophoresis. Lanes 1 and 8 labeled as ‘C’ are control lanes having untreated DNA.

    Journal: Nucleic Acids Research

    Article Title: Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4

    doi: 10.1093/nar/gkq1191

    Figure Lengend Snippet: ATP stimulates gp17 nuclease. ( A ) Nuclease activity of gp17 is stimulated in presence of ATP. gp17 (1 µM) was incubated with linear pAd10 DNA (100 ng) in the presence of increasing concentrations of ATP (0.05–5 mM) . Note that the DNA is degraded to small fragments in some of the lanes. Therefore, very little DNA smear is seen in these lanes. ( B ) Nuclease activity of gp17 in the presence of ATP analogs. gp17 (1.2 µM) was incubated with circular pAd10 DNA (200 ng) in the presence of ATP, ADP, ATP-γS or AMP-PNP (1 mM). ( C ) The T4 nuclease domain (C360, amino acids 360–577) (left panel; lanes 1–7) or the RB49 nuclease domain (C360, amino acids 358–607) (right panel; lanes 8–14) is not stimulated by ATP. T4 C360 (4 µM) or RB49 C360 (1 µM), either alone (lanes 2 and 9) or in the presence of ATP (lanes 3–7 and 10–14) was incubated with linear pAd10 DNA (100 ng) for 15 min and the samples were analyzed by 0.8% (w/v) agarose gel electrophoresis. Lanes 1 and 8 labeled as ‘C’ are control lanes having untreated DNA.

    Article Snippet: The digested DNA was precipitated by 8 M ammonium acetate and resuspended in water, followed by the addition of T4 DNA ligase (Fermentas) or E. coli DNA ligase (New England Biolabs) in a 25 µl reaction mixture.

    Techniques: Activity Assay, Incubation, Agarose Gel Electrophoresis, Labeling

    gp17 nuclease prefers long DNA substrates and cleaves at the ends of linear DNA. ( A ) Increasing concentrations of gp17 were incubated with 0.9 nM each of 29 kb pAd10 plasmid DNA or 2.6 kb pUC19 plasmid DNA. The undigested circular DNA was quantified and used to determine the percent of cleaved DNA at different gp17:DNA ratios. Values represent the average of duplicates from two independent experiments. ( B ) gp17 preference for longer DNA molecules was seen by incubating gp17 (3 µM, lanes 2–7) with a 2-log DNA ladder (400 ng, 0.1–10 kb, New England Biolabs) for 2–30 min. ( C ) Autoradiogram showing the cleavage of γ 32 P end-labeled λ-HindIII DNA fragments (0.5 pmol, 125–23 130 bp, Promega) by gp17 (1.2 µM) (lanes 2–6) or DNase I (0.0024 µM, 500-fold less than gp17) (lanes 7–11). Lane 1 has untreated DNA. ( D ) gp17 nuclease generates blunt ends. Circular pUC19 DNA (40 ng) was cleaved by gp17 (lanes 2–4) or BamH1 (lanes 5–7). The cleaved DNA was then treated with E. coli DNA ligase (lanes 3 and 6) or T4 DNA ligase (lanes 4 and 7). Lanes labeled as ‘C’ are control untreated lanes. See ‘Materials and Methods’ section for additional details.

    Journal: Nucleic Acids Research

    Article Title: Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4

    doi: 10.1093/nar/gkq1191

    Figure Lengend Snippet: gp17 nuclease prefers long DNA substrates and cleaves at the ends of linear DNA. ( A ) Increasing concentrations of gp17 were incubated with 0.9 nM each of 29 kb pAd10 plasmid DNA or 2.6 kb pUC19 plasmid DNA. The undigested circular DNA was quantified and used to determine the percent of cleaved DNA at different gp17:DNA ratios. Values represent the average of duplicates from two independent experiments. ( B ) gp17 preference for longer DNA molecules was seen by incubating gp17 (3 µM, lanes 2–7) with a 2-log DNA ladder (400 ng, 0.1–10 kb, New England Biolabs) for 2–30 min. ( C ) Autoradiogram showing the cleavage of γ 32 P end-labeled λ-HindIII DNA fragments (0.5 pmol, 125–23 130 bp, Promega) by gp17 (1.2 µM) (lanes 2–6) or DNase I (0.0024 µM, 500-fold less than gp17) (lanes 7–11). Lane 1 has untreated DNA. ( D ) gp17 nuclease generates blunt ends. Circular pUC19 DNA (40 ng) was cleaved by gp17 (lanes 2–4) or BamH1 (lanes 5–7). The cleaved DNA was then treated with E. coli DNA ligase (lanes 3 and 6) or T4 DNA ligase (lanes 4 and 7). Lanes labeled as ‘C’ are control untreated lanes. See ‘Materials and Methods’ section for additional details.

    Article Snippet: The digested DNA was precipitated by 8 M ammonium acetate and resuspended in water, followed by the addition of T4 DNA ligase (Fermentas) or E. coli DNA ligase (New England Biolabs) in a 25 µl reaction mixture.

    Techniques: Incubation, Plasmid Preparation, Labeling

    Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, T4 ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).

    Journal: Nucleic Acids Research

    Article Title: Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro

    doi:

    Figure Lengend Snippet: Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, T4 ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).

    Article Snippet: The reaction was incubated for 10 min at 37°C in T4 ligase buffer (New England Biolabs) containing 100 µg/ml bovine serum albumin, 75 mM KCl and the heteroduplex oligo recovered after Dpn II digestion (estimated to be a ∼100-fold molar excess over the plasmid ends).

    Techniques: Plasmid Preparation, Ligation, Generated

    T4 DNA-ligase activity in the presence of SA. T4 DNA-ligase was treated or not with increasing SA concentrations (15 min at 25°C) and then incubated at 37°C for 1 min with the oligo substrate. ( A ) The oligo(dT) 16  multimers were separated in polyacrylamide/urea gels: T4 DNA-ligase without SA (lane 1) or incubated with increasing SA concentrations of 2.5(2), 5(3), 10(4), and 20(5) μM. ( B ) The activity was quantitated using an InstantImager (Packard). ( C ) Inhibition of enzyme-adenylate formation by SA. T4 DNA-ligase was incubated or not with increasing SA concentrations (15 min at 25°C) before the addition of [α- 32 P]ATP. The enzyme adenylate complexes were separated by electrophoresis and detected by autoradiography.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Deficient DNA-ligase activity in the metabolic disease tyrosinemia type I

    doi:

    Figure Lengend Snippet: T4 DNA-ligase activity in the presence of SA. T4 DNA-ligase was treated or not with increasing SA concentrations (15 min at 25°C) and then incubated at 37°C for 1 min with the oligo substrate. ( A ) The oligo(dT) 16 multimers were separated in polyacrylamide/urea gels: T4 DNA-ligase without SA (lane 1) or incubated with increasing SA concentrations of 2.5(2), 5(3), 10(4), and 20(5) μM. ( B ) The activity was quantitated using an InstantImager (Packard). ( C ) Inhibition of enzyme-adenylate formation by SA. T4 DNA-ligase was incubated or not with increasing SA concentrations (15 min at 25°C) before the addition of [α- 32 P]ATP. The enzyme adenylate complexes were separated by electrophoresis and detected by autoradiography.

    Article Snippet: T4 DNA-ligase (2 units, 0.150 μg of proteins) was incubated (final volume, 10 μl) in a buffer containing 66 mM Hepes at pH 8.0, 10 mM MgCl2 , and increasing SA concentrations for 15 min at 25°C.

    Techniques: Activity Assay, Incubation, Inhibition, Electrophoresis, Autoradiography

    SCIPay design permits CRISPR-guided integration of specific donor DNA sequences via homology directed repair. (A) Overview of SCIPay design. Here, HA present in the payload sequence lead to its genomic integration via HDR. (B – G) EL4 cells were electroporated with SCIPay containing a BFP transgene targeting B2M with various HA lengths. (B) Percentage of knock-out cells. (C) Efficiency and (D) precision scores for GFP, denoting integration of the non-payload plasmid sequence. (E) Efficiency and (F) precision scores for BFP, denoting HDR integration of BFP. (G) Ratio of BFP-to GFP-positive cells. (H) Representative flow cytometry plots. Results in (B – G) represent means +/- SEM of 4 independent experiments. Data was analyzed using 1-way ANOVA and significant differences are indicated with lower case letters, where groups with different letters are significantly different than each other (p

    Journal: bioRxiv

    Article Title: Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of large genetic payloads for rapid cell engineering

    doi: 10.1101/2020.03.25.008276

    Figure Lengend Snippet: SCIPay design permits CRISPR-guided integration of specific donor DNA sequences via homology directed repair. (A) Overview of SCIPay design. Here, HA present in the payload sequence lead to its genomic integration via HDR. (B – G) EL4 cells were electroporated with SCIPay containing a BFP transgene targeting B2M with various HA lengths. (B) Percentage of knock-out cells. (C) Efficiency and (D) precision scores for GFP, denoting integration of the non-payload plasmid sequence. (E) Efficiency and (F) precision scores for BFP, denoting HDR integration of BFP. (G) Ratio of BFP-to GFP-positive cells. (H) Representative flow cytometry plots. Results in (B – G) represent means +/- SEM of 4 independent experiments. Data was analyzed using 1-way ANOVA and significant differences are indicated with lower case letters, where groups with different letters are significantly different than each other (p

    Article Snippet: This modular plasmid allows insertion of gRNA-coding DNA oligonucleotides using BpiI as outlined above and subsequent cloning of HA- and bait-coding DNA fragments engineered to be in-frame with the preceding genome sequence and following P2A-BFP sequence with BsaI using Gibson or restriction/ligation cloning.

    Techniques: CRISPR, Sequencing, Knock-Out, Plasmid Preparation, Flow Cytometry

    3′ Branch ligation by T4 DNA ligase at non-conventional DNA ends formed by nicks, gaps, and overhangs. (a) Schematic representation of ligation assay with different DNA accepter types. The blunt-end DNA donor (blue) is a synthetic, partially dsDNA molecule with dideoxy 3 ′ -termini (filled circles) to prevent DNA donor self-ligation. The long arm of the donor is 5 ′- phosporylated. The DNA acceptors were assembled using 2 or 3 oligos (black, red, and orange lines) to form a nick (without phosphates), a gap (1 or 8 nt), or a 36-nt 3 ′ -recessive end. All strands of the substrates are unphosphorylated, and the scaffold strand is 3 ′ dideoxy protected. (b) Analysis of the size shift of ligated products of substrates 1, 2, 3, and 4, respectively, using a 6% denaturing polyacrylamide gel. Reactions were performed according to the optimized condition. The negative no-ligase controls (lanes 1, 3, 4, 6, 7, 9, 10, 12, and 13) were loaded at 1 or 0.5× volume of corresponding experimental assays. If ligation occurs, the substrate size is shifted up by 22 nt. Red arrowheads correspond to the substrate, and purple arrowheads correspond to donor-ligated substrates. Donor and substrate sequences in Supplementary Table S1 . (c) Expected sizes of substrate and ligation product and approximate ligation efficiency in each experimental group. The intensity of each band was estimated using ImageJ and normalized by its expected size. Ligation efficiency was estimated by dividing the normalized intensity of ligated products by the normalized total intensity of ligated and unligated products.

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: 3′ Branch ligation: a novel method to ligate non-complementary DNA to recessed or internal 3′OH ends in DNA or RNA

    doi: 10.1093/dnares/dsy037

    Figure Lengend Snippet: 3′ Branch ligation by T4 DNA ligase at non-conventional DNA ends formed by nicks, gaps, and overhangs. (a) Schematic representation of ligation assay with different DNA accepter types. The blunt-end DNA donor (blue) is a synthetic, partially dsDNA molecule with dideoxy 3 ′ -termini (filled circles) to prevent DNA donor self-ligation. The long arm of the donor is 5 ′- phosporylated. The DNA acceptors were assembled using 2 or 3 oligos (black, red, and orange lines) to form a nick (without phosphates), a gap (1 or 8 nt), or a 36-nt 3 ′ -recessive end. All strands of the substrates are unphosphorylated, and the scaffold strand is 3 ′ dideoxy protected. (b) Analysis of the size shift of ligated products of substrates 1, 2, 3, and 4, respectively, using a 6% denaturing polyacrylamide gel. Reactions were performed according to the optimized condition. The negative no-ligase controls (lanes 1, 3, 4, 6, 7, 9, 10, 12, and 13) were loaded at 1 or 0.5× volume of corresponding experimental assays. If ligation occurs, the substrate size is shifted up by 22 nt. Red arrowheads correspond to the substrate, and purple arrowheads correspond to donor-ligated substrates. Donor and substrate sequences in Supplementary Table S1 . (c) Expected sizes of substrate and ligation product and approximate ligation efficiency in each experimental group. The intensity of each band was estimated using ImageJ and normalized by its expected size. Ligation efficiency was estimated by dividing the normalized intensity of ligated products by the normalized total intensity of ligated and unligated products.

    Article Snippet: A previous study reported that for sealing nicks in DNA/RNA hybrids, T4 DNA ligase, and T4 RNA ligase 2, but not T4 RNA ligase 1, can effectively join a 5 ′ PO4 DNA end to a juxtaposed 3 ′ OH DNA or RNA end when the complimentary strand is RNA but not DNA.

    Techniques: Ligation

    3′ Branch ligation at the 3′ end of RNA in DNA/RNA hybrid. Schematic representation of 3′-branch ligation on a DNA/RNA hybrid with a 20‐bp complimentary region. We tested whether blunt-end DNA donors would ligate to the 3 ′ -recessive end of DNA and/or to the 3 ′ -recessive end of RNA. DNA(ON-21) hybridizes with the RNA strand (a), whereas DNA(ON-23) cannot hybridize with the RNA strand (b). (c, d) Gel analysis of size shift of ligated products using 6% denaturing polyacrylamide gel. The red arrowheads correspond to the RNA substrate (29 nt), and the green arrowhead corresponds to DNA substrate (80 nt). The purple arrowhead corresponds to donor-ligated RNA substrates. If ligation occurs, the substrate size would shift up by 20 nt. (c) Lanes 1 and 2, experimental duplicates; lanes 7–10, no-ligase controls; 10% PEG was added with T4 DNA ligase. (d) Lane 1, no-ligase control; lanes 2, 3, and 8, T4 DNA ligase with 10% PEG; lanes 4, 5, and 9, T4 RNA ligase 1 with 20% DMSO; lanes 6, 7, and 10, T4 RNA ligase 2 with 20% DMSO.

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: 3′ Branch ligation: a novel method to ligate non-complementary DNA to recessed or internal 3′OH ends in DNA or RNA

    doi: 10.1093/dnares/dsy037

    Figure Lengend Snippet: 3′ Branch ligation at the 3′ end of RNA in DNA/RNA hybrid. Schematic representation of 3′-branch ligation on a DNA/RNA hybrid with a 20‐bp complimentary region. We tested whether blunt-end DNA donors would ligate to the 3 ′ -recessive end of DNA and/or to the 3 ′ -recessive end of RNA. DNA(ON-21) hybridizes with the RNA strand (a), whereas DNA(ON-23) cannot hybridize with the RNA strand (b). (c, d) Gel analysis of size shift of ligated products using 6% denaturing polyacrylamide gel. The red arrowheads correspond to the RNA substrate (29 nt), and the green arrowhead corresponds to DNA substrate (80 nt). The purple arrowhead corresponds to donor-ligated RNA substrates. If ligation occurs, the substrate size would shift up by 20 nt. (c) Lanes 1 and 2, experimental duplicates; lanes 7–10, no-ligase controls; 10% PEG was added with T4 DNA ligase. (d) Lane 1, no-ligase control; lanes 2, 3, and 8, T4 DNA ligase with 10% PEG; lanes 4, 5, and 9, T4 RNA ligase 1 with 20% DMSO; lanes 6, 7, and 10, T4 RNA ligase 2 with 20% DMSO.

    Article Snippet: A previous study reported that for sealing nicks in DNA/RNA hybrids, T4 DNA ligase, and T4 RNA ligase 2, but not T4 RNA ligase 1, can effectively join a 5 ′ PO4 DNA end to a juxtaposed 3 ′ OH DNA or RNA end when the complimentary strand is RNA but not DNA.

    Techniques: Ligation

    Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard T4 DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.

    Journal: Nucleic Acids Research

    Article Title: SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing

    doi: 10.1093/nar/gkw1110

    Figure Lengend Snippet: Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard T4 DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.

    Article Snippet: For the 3΄-end ligation; adapter ss1 (final conc 10 μM), T4 DNA ligase buffer (40 mM Tris–HCl pH 7.8,10 mM MgCl2 , 10 mM DTT, 0.5 mM ATP), PEG4000 (5% w/v) and 30 units T4 DNA ligase (Thermo Fisher Scientific) and nuclease free water was added to the sample on ice, in a total volume of 30 μl.

    Techniques: Bisulfite Sequencing, Methylation, Construct, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing, Sequencing, DNA Methylation Assay, Random Hexamer Labeling, DNA Ligation, Modification, Ligation

    RNAse protection assay. The upper panel shows a schematic representation of part of the 5′ region of the human CD45 gene and the RNA probes generated in vitro . The lower panel shows the protected fragments in different cell lines: Jurkat (human T cell), Raji (human B cell), K562 (human erythroid), U937 (human myeloid), EL-4 (mouse T cell) and HFB-1 (human B cell). M is the Hin fI-digested φX174 DNA used as a marker. The size of the bands in bp is indicated at both sides.

    Journal: Immunology

    Article Title: Structural and functional analysis of the human CD45 gene (PTPRC) upstream region: evidence for a functional promoter within the first intron of the gene

    doi: 10.1046/j.1365-2567.2001.01177.x

    Figure Lengend Snippet: RNAse protection assay. The upper panel shows a schematic representation of part of the 5′ region of the human CD45 gene and the RNA probes generated in vitro . The lower panel shows the protected fragments in different cell lines: Jurkat (human T cell), Raji (human B cell), K562 (human erythroid), U937 (human myeloid), EL-4 (mouse T cell) and HFB-1 (human B cell). M is the Hin fI-digested φX174 DNA used as a marker. The size of the bands in bp is indicated at both sides.

    Article Snippet: Second strand was generated by incubating the first-strand product with DNA polymerase I in the presence of RNase H and DNA ligase (all from Life Technologies) for 2 hr at 16°.

    Techniques: Rnase Protection Assay, Generated, In Vitro, Marker

    Detection of stage-specific RNAs and the corresponding macronuclear genes. Total RNA of vegetative cells and different stages during macronuclear development was isolated, first and second strand cDNA synthesis was performed (see Materials and Methods). For agarose gel electrophoresis equal volumes from the same cDNA preparation were used for each of the four gels. Lane 1, cDNA derived from vegetative cells; lanes 2–5, cDNA derived from exconjugants, 0, 20, 30 or 40 h PC, respectively. The gels were blotted and hybridized with Dig labeled probes generated by PCR amplification of the differentially expressed clones mdp1 ( A ), mdp2 ( B ) and mdp3 ( C ). To check the integrity of the different cDNAs, actin I was used as a control ( D ). Macronuclear DNA was isolated and separated by agarose gel electrophoresis. The gels were blotted and hybridized with the corresponding Dig labeled probes (lane 6).

    Journal: Nucleic Acids Research

    Article Title: A PIWI homolog is one of the proteins expressed exclusively during macronuclear development in the ciliate Stylonychia lemnae

    doi:

    Figure Lengend Snippet: Detection of stage-specific RNAs and the corresponding macronuclear genes. Total RNA of vegetative cells and different stages during macronuclear development was isolated, first and second strand cDNA synthesis was performed (see Materials and Methods). For agarose gel electrophoresis equal volumes from the same cDNA preparation were used for each of the four gels. Lane 1, cDNA derived from vegetative cells; lanes 2–5, cDNA derived from exconjugants, 0, 20, 30 or 40 h PC, respectively. The gels were blotted and hybridized with Dig labeled probes generated by PCR amplification of the differentially expressed clones mdp1 ( A ), mdp2 ( B ) and mdp3 ( C ). To check the integrity of the different cDNAs, actin I was used as a control ( D ). Macronuclear DNA was isolated and separated by agarose gel electrophoresis. The gels were blotted and hybridized with the corresponding Dig labeled probes (lane 6).

    Article Snippet: Total RNA (3 µg) was reverse transcribed and second strand cDNA synthesis was carried out with Escherichia coli DNA polymerase I, E.coli RNase H and E.coli DNA Ligase (SuperScript™ Choice System for cDNA Synthesis, Invitrogen).

    Techniques: Isolation, Agarose Gel Electrophoresis, Derivative Assay, Labeling, Generated, Polymerase Chain Reaction, Amplification, Clone Assay

    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).

    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

    Effect of DNA ligase addition on the DNA ligase-[α- 32 P]AMP reduction. ( A ) Cell-free DNA repair assay was carried out for 60 min with HeLa S3 cell extracts containing DNA ligase-[α- 32 P]AMP. Plasmid DNA containing either γ-ray-induced SSIs (γ-SSI) or alkylated base damage induced by MNNG was used for the assay. The reactions also contained Lig I, Lig III or T4 DNA ligase (T4 Lig). After the repair reaction, the mixtures were fractionated on an SDS–7.5% polyacrylamide gel, and 32 P activity was visualized by autoradiography. Alternatively, 32 P activities were measured with an AlphaImager (Packard) and quantified results are shown in ( B ) (γ-SSI) and ( C ) (MNNG). The results shown are from one of four independent experiments. The amount of Lig I-[ 32 P]AMP (non-damaged DNA) was calculated as 100%.

    Journal: Nucleic Acids Research

    Article Title: Repair of single-strand DNA interruptions by redundant pathways and its implication in cellular sensitivity to DNA-damaging agents

    doi: 10.1093/nar/gkg892

    Figure Lengend Snippet: Effect of DNA ligase addition on the DNA ligase-[α- 32 P]AMP reduction. ( A ) Cell-free DNA repair assay was carried out for 60 min with HeLa S3 cell extracts containing DNA ligase-[α- 32 P]AMP. Plasmid DNA containing either γ-ray-induced SSIs (γ-SSI) or alkylated base damage induced by MNNG was used for the assay. The reactions also contained Lig I, Lig III or T4 DNA ligase (T4 Lig). After the repair reaction, the mixtures were fractionated on an SDS–7.5% polyacrylamide gel, and 32 P activity was visualized by autoradiography. Alternatively, 32 P activities were measured with an AlphaImager (Packard) and quantified results are shown in ( B ) (γ-SSI) and ( C ) (MNNG). The results shown are from one of four independent experiments. The amount of Lig I-[ 32 P]AMP (non-damaged DNA) was calculated as 100%.

    Article Snippet: The activity of the resulting DNA ligases was determined using T4 DNA ligase (Amersham Biosciences) as a standard and expressed in Weiss units.

    Techniques: Plasmid Preparation, Activity Assay, Autoradiography

    Assay design and preparation of cell-free extracts containing DNA ligase-[α- 32 P]AMP. ( A ) Whole cell-free extracts were treated with PPi to remove AMP from DNA ligases. PPi was then dialyzed out and the dialyzed extracts were incubated with [α- 32 P]ATP to form DNA ligase- [α- 32 P]AMP. Extracts containing DNA ligase-[α- 32 P]AMP were used for cell-free DNA repair assays. When the DNA ligase-[α- 32 P]AMP is used to rejoin DNA nicks, [α- 32 P]AMP is released from the DNA ligase. ( B ) Cell-free extracts containing DNA ligase-[α- 32 P]AMP, recombinant Lig I- [α- 32 P]AMP, Lig III-[α- 32 P]AMP and T4 DNA ligase (T4 Lig)-[α- 32 P]AMP were fractionated by SDS–7.5% PAGE and 32 P activity was visualized by autoradiography.

    Journal: Nucleic Acids Research

    Article Title: Repair of single-strand DNA interruptions by redundant pathways and its implication in cellular sensitivity to DNA-damaging agents

    doi: 10.1093/nar/gkg892

    Figure Lengend Snippet: Assay design and preparation of cell-free extracts containing DNA ligase-[α- 32 P]AMP. ( A ) Whole cell-free extracts were treated with PPi to remove AMP from DNA ligases. PPi was then dialyzed out and the dialyzed extracts were incubated with [α- 32 P]ATP to form DNA ligase- [α- 32 P]AMP. Extracts containing DNA ligase-[α- 32 P]AMP were used for cell-free DNA repair assays. When the DNA ligase-[α- 32 P]AMP is used to rejoin DNA nicks, [α- 32 P]AMP is released from the DNA ligase. ( B ) Cell-free extracts containing DNA ligase-[α- 32 P]AMP, recombinant Lig I- [α- 32 P]AMP, Lig III-[α- 32 P]AMP and T4 DNA ligase (T4 Lig)-[α- 32 P]AMP were fractionated by SDS–7.5% PAGE and 32 P activity was visualized by autoradiography.

    Article Snippet: The activity of the resulting DNA ligases was determined using T4 DNA ligase (Amersham Biosciences) as a standard and expressed in Weiss units.

    Techniques: Incubation, Recombinant, Polyacrylamide Gel Electrophoresis, Activity Assay, Autoradiography