t4 dna ligase  (New England Biolabs)


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    Structured Review

    New England Biolabs t4 dna ligase
    FEN1 completes primer removal and L-strand maturation. ( A ) Schematic of template used in panel B. The possible products are illustrated (b–d). ( B ) Coupled nuclease gap-filling ligation assay was performed as in Figure 3 but in the presence of the indicated nucleases. MGME1 (250 fmol) was added in lanes 4 and 9; FEN1 (35 fmol) was added in lanes 5 and 10. The samples in lanes 6–10 contained 300 fmol DNA ligase III. Conversion of the nicked product (c) to a ligated 80 nt product (d) was stimulated when FEN1 was added together with RNase H1. ( C ) Schematic of template used in panel D. The downstream chimeric oligonucleotide was labelled at the 3′-end. The possible products are illustrated (b–d). ( D ) Coupled nuclease gap-filling ligation assay was performed in the presence of RNase H1 alone or together with FEN1 (35 fmol) as indicated. Lanes 2–4 contained only RNase H1 with or without FEN1 to monitor nuclease activity. RNase H1 cleaved the downstream oligonucleotide leaving 1–3 unprocessed ribonucleotides. FEN1 cleaved the remaining ribonucleotides, resulting in a shorter product. POLγ and DNA ligase (ligase III or <t>T4</t> DNA ligase) were added in lanes 5–10. Ligase III showed ligation only when both RNase H1 and FEN1 were added. However, T4 DNA ligase could ligate without FEN1. Lanes 12–14 are ligation controls using a 3′-end labelled DNA-only oligonucleotide with a 5′-end phosphate. Note that the short band (
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL"

    Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky708

    FEN1 completes primer removal and L-strand maturation. ( A ) Schematic of template used in panel B. The possible products are illustrated (b–d). ( B ) Coupled nuclease gap-filling ligation assay was performed as in Figure 3 but in the presence of the indicated nucleases. MGME1 (250 fmol) was added in lanes 4 and 9; FEN1 (35 fmol) was added in lanes 5 and 10. The samples in lanes 6–10 contained 300 fmol DNA ligase III. Conversion of the nicked product (c) to a ligated 80 nt product (d) was stimulated when FEN1 was added together with RNase H1. ( C ) Schematic of template used in panel D. The downstream chimeric oligonucleotide was labelled at the 3′-end. The possible products are illustrated (b–d). ( D ) Coupled nuclease gap-filling ligation assay was performed in the presence of RNase H1 alone or together with FEN1 (35 fmol) as indicated. Lanes 2–4 contained only RNase H1 with or without FEN1 to monitor nuclease activity. RNase H1 cleaved the downstream oligonucleotide leaving 1–3 unprocessed ribonucleotides. FEN1 cleaved the remaining ribonucleotides, resulting in a shorter product. POLγ and DNA ligase (ligase III or T4 DNA ligase) were added in lanes 5–10. Ligase III showed ligation only when both RNase H1 and FEN1 were added. However, T4 DNA ligase could ligate without FEN1. Lanes 12–14 are ligation controls using a 3′-end labelled DNA-only oligonucleotide with a 5′-end phosphate. Note that the short band (
    Figure Legend Snippet: FEN1 completes primer removal and L-strand maturation. ( A ) Schematic of template used in panel B. The possible products are illustrated (b–d). ( B ) Coupled nuclease gap-filling ligation assay was performed as in Figure 3 but in the presence of the indicated nucleases. MGME1 (250 fmol) was added in lanes 4 and 9; FEN1 (35 fmol) was added in lanes 5 and 10. The samples in lanes 6–10 contained 300 fmol DNA ligase III. Conversion of the nicked product (c) to a ligated 80 nt product (d) was stimulated when FEN1 was added together with RNase H1. ( C ) Schematic of template used in panel D. The downstream chimeric oligonucleotide was labelled at the 3′-end. The possible products are illustrated (b–d). ( D ) Coupled nuclease gap-filling ligation assay was performed in the presence of RNase H1 alone or together with FEN1 (35 fmol) as indicated. Lanes 2–4 contained only RNase H1 with or without FEN1 to monitor nuclease activity. RNase H1 cleaved the downstream oligonucleotide leaving 1–3 unprocessed ribonucleotides. FEN1 cleaved the remaining ribonucleotides, resulting in a shorter product. POLγ and DNA ligase (ligase III or T4 DNA ligase) were added in lanes 5–10. Ligase III showed ligation only when both RNase H1 and FEN1 were added. However, T4 DNA ligase could ligate without FEN1. Lanes 12–14 are ligation controls using a 3′-end labelled DNA-only oligonucleotide with a 5′-end phosphate. Note that the short band (

    Techniques Used: Ligation, Activity Assay

    RNase H1 processing coupled to POLγ dependent DNA synthesis does not produce ligatable nicks. ( A ) Schematic of the coupled nuclease gap-filling ligation assay performed on a gapped OriL substrate (a). The upstream oligonucleotide was radioactively labelled at the 5′-end. The possible products are illustrated (b–d). (–) Coupled nuclease gap-filling ligation assay as shown in A. POLγ filled the gap (lane 2, marked b) and had limited strand displacement activity (SD). Note though that POLγ completely displaces the downstream oligonucleotide in a small fraction of templates (80 nt band, lanes 3–6,), RNase H1 cleaved the RNA in the substrate (lane 3), enabling further gap-filling (marked c). Only very low levels of ligated products were formed in the presence of 80–320 fmol DNA ligase III (lanes 4–6). A prominent 80 nt ligated product was formed with T4 DNA ligase (lane 7, marked d). The letters a-d correspond to the illustrations in panel A. ( C ) Ligation assay on a nicked substrate containing RNA tracts of varying length downstream of the nick in the presence of 80–320 fmol DNA ligase III. DNA ligase III discriminates against nicked substrates that contain increasing stretches of ribonucleotides. Two, but not five or more ribonucleotides, can be ligated. ( D ) As in C, except performed with T4 ligase (1–8 U). T4 ligase can ligate five but not 10 ribonucleotides. ( E ) T4 ligase-mediated ligation is abolished in the presence of the mutant RNase H1 proteins. The letters a-d correspond to the illustrations in panel A.
    Figure Legend Snippet: RNase H1 processing coupled to POLγ dependent DNA synthesis does not produce ligatable nicks. ( A ) Schematic of the coupled nuclease gap-filling ligation assay performed on a gapped OriL substrate (a). The upstream oligonucleotide was radioactively labelled at the 5′-end. The possible products are illustrated (b–d). (–) Coupled nuclease gap-filling ligation assay as shown in A. POLγ filled the gap (lane 2, marked b) and had limited strand displacement activity (SD). Note though that POLγ completely displaces the downstream oligonucleotide in a small fraction of templates (80 nt band, lanes 3–6,), RNase H1 cleaved the RNA in the substrate (lane 3), enabling further gap-filling (marked c). Only very low levels of ligated products were formed in the presence of 80–320 fmol DNA ligase III (lanes 4–6). A prominent 80 nt ligated product was formed with T4 DNA ligase (lane 7, marked d). The letters a-d correspond to the illustrations in panel A. ( C ) Ligation assay on a nicked substrate containing RNA tracts of varying length downstream of the nick in the presence of 80–320 fmol DNA ligase III. DNA ligase III discriminates against nicked substrates that contain increasing stretches of ribonucleotides. Two, but not five or more ribonucleotides, can be ligated. ( D ) As in C, except performed with T4 ligase (1–8 U). T4 ligase can ligate five but not 10 ribonucleotides. ( E ) T4 ligase-mediated ligation is abolished in the presence of the mutant RNase H1 proteins. The letters a-d correspond to the illustrations in panel A.

    Techniques Used: DNA Synthesis, Ligation, Activity Assay, Mutagenesis

    2) Product Images from "Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research"

    Article Title: Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research

    Journal: Plants

    doi: 10.3390/plants9091090

    Schematic diagram of the nicking endonucleases-mediated DNA assembly (NEMDA) strategy for plant ihpRNA vector constructions. ( A ) The pRNAi-NE includes the 35S CaMV promoter, the Catalase intron, the ccdB gene, and four Nb.BtvCI and Xba I recognition sites with differently designed adaptors (different colors). ( B ) The sense and antisense PCR products have four Nb.BtsI recognition sites with differently designed adaptors (different colors). ( C ) One-step construction of an ihpRNA vector. The target fragments of the gene of interest are PCR amplified using gene-specific primers carrying Nb.BtsI sites and adaptors complementary to the appropriate sequences on the vector. The unpurified PCR products digested by Nb.BtsI are mixed, in one tube, with unpurified pRNAi-NE vector digested by Nb.BtvCI and Xba I, for heat-inactivation of these restriction endonucleases and melting out of the nicked end strands, annealing. The T4 DNA ligase also can be used to increase cloning efficiency. The reaction product is transferred into E. coli competent cells to produce the pRNAi plasmid ( D ).
    Figure Legend Snippet: Schematic diagram of the nicking endonucleases-mediated DNA assembly (NEMDA) strategy for plant ihpRNA vector constructions. ( A ) The pRNAi-NE includes the 35S CaMV promoter, the Catalase intron, the ccdB gene, and four Nb.BtvCI and Xba I recognition sites with differently designed adaptors (different colors). ( B ) The sense and antisense PCR products have four Nb.BtsI recognition sites with differently designed adaptors (different colors). ( C ) One-step construction of an ihpRNA vector. The target fragments of the gene of interest are PCR amplified using gene-specific primers carrying Nb.BtsI sites and adaptors complementary to the appropriate sequences on the vector. The unpurified PCR products digested by Nb.BtsI are mixed, in one tube, with unpurified pRNAi-NE vector digested by Nb.BtvCI and Xba I, for heat-inactivation of these restriction endonucleases and melting out of the nicked end strands, annealing. The T4 DNA ligase also can be used to increase cloning efficiency. The reaction product is transferred into E. coli competent cells to produce the pRNAi plasmid ( D ).

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Clone Assay

    3) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.284992

    Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and
    Figure Legend Snippet: Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and

    Techniques Used:

    Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .
    Figure Legend Snippet: Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .

    Techniques Used: Diffusion-based Assay, Binding Assay

    Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (
    Figure Legend Snippet: Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (

    Techniques Used:

    4) Product Images from "The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends"

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp695

    Interaction of HMO2 with plasmid DNA. ( A , B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.
    Figure Legend Snippet: Interaction of HMO2 with plasmid DNA. ( A , B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis

    HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.
    Figure Legend Snippet: HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.

    Techniques Used: Ligation

    HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.
    Figure Legend Snippet: HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.

    Techniques Used:

    DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM B. subtilis HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM B. subtilis HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.
    Figure Legend Snippet: DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM B. subtilis HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM B. subtilis HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.

    Techniques Used: Sequencing, Incubation, Ligation

    5) Product Images from "The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends"

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp695

    Interaction of HMO2 with plasmid DNA. ( A , B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.
    Figure Legend Snippet: Interaction of HMO2 with plasmid DNA. ( A , B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis

    HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.
    Figure Legend Snippet: HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.

    Techniques Used: Ligation

    HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.
    Figure Legend Snippet: HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.

    Techniques Used:

    DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM B. subtilis HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM B. subtilis HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.
    Figure Legend Snippet: DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM B. subtilis HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM B. subtilis HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.

    Techniques Used: Sequencing, Incubation, Ligation

    6) Product Images from "The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends"

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp695

    Interaction of HMO2 with plasmid DNA. ( A , B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.
    Figure Legend Snippet: Interaction of HMO2 with plasmid DNA. ( A , B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis

    HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.
    Figure Legend Snippet: HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.

    Techniques Used: Ligation

    HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.
    Figure Legend Snippet: HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.

    Techniques Used:

    DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM B. subtilis HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM B. subtilis HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.
    Figure Legend Snippet: DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM B. subtilis HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM B. subtilis HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.

    Techniques Used: Sequencing, Incubation, Ligation

    7) Product Images from "DNA bending, compaction and negative supercoiling by the architectural protein Sso7d of Sulfolobus solfataricus"

    Article Title: DNA bending, compaction and negative supercoiling by the architectural protein Sso7d of Sulfolobus solfataricus

    Journal: Nucleic Acids Research

    doi:

    Ring-closure assay. Each lane contained 1 ng (0.6 nM) of end-labelled 129 bp DNA fragment. Each pair of lanes shows replicated independent experiments. Lanes 1 and 2, the input fragment; lanes 3 and 4, the fragment incubated with T4 DNA ligase. In the other lanes the fragment was incubated, before ligation, with: lanes 5 and 6, 600 ng (2 µM) Lrs14; lanes 7 and 8, 300 ng (2 µM) Sso7d; lanes 9 and 10, 300 ng (2 µM) HMfA. The protein/DNA ratio was 26 molecules/bp for all proteins.
    Figure Legend Snippet: Ring-closure assay. Each lane contained 1 ng (0.6 nM) of end-labelled 129 bp DNA fragment. Each pair of lanes shows replicated independent experiments. Lanes 1 and 2, the input fragment; lanes 3 and 4, the fragment incubated with T4 DNA ligase. In the other lanes the fragment was incubated, before ligation, with: lanes 5 and 6, 600 ng (2 µM) Lrs14; lanes 7 and 8, 300 ng (2 µM) Sso7d; lanes 9 and 10, 300 ng (2 µM) HMfA. The protein/DNA ratio was 26 molecules/bp for all proteins.

    Techniques Used: Incubation, Ligation

    8) Product Images from "Analysis of RNA 5′ ends: phosphate enumeration and cap characterization"

    Article Title: Analysis of RNA 5′ ends: phosphate enumeration and cap characterization

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2018.10.023

    Detection of monophosphorylated 5′ ends by PABLO PABLO is a splinted ligation assay in which monophosphorylated RNA 5′ ends are selectively ligated to the 3′ end of a DNA oligonucleotide (oligo X) when the two are juxtaposed by simultaneous base pairing to a bridging DNA oligonucleotide (oligo Y) and treated with T4 DNA ligase. The ligation product and its unligated counterpart are then separated by electrophoresis on a denaturing gel and detected by Northern blotting. The differential mobility of these two bands can be enhanced by site-specific RNA cleavage with a DNAzyme (not shown). TriP, triphosphorylated RNA; DiP, diphosphorylated RNA; MonoP, monophosphorylated RNA.
    Figure Legend Snippet: Detection of monophosphorylated 5′ ends by PABLO PABLO is a splinted ligation assay in which monophosphorylated RNA 5′ ends are selectively ligated to the 3′ end of a DNA oligonucleotide (oligo X) when the two are juxtaposed by simultaneous base pairing to a bridging DNA oligonucleotide (oligo Y) and treated with T4 DNA ligase. The ligation product and its unligated counterpart are then separated by electrophoresis on a denaturing gel and detected by Northern blotting. The differential mobility of these two bands can be enhanced by site-specific RNA cleavage with a DNAzyme (not shown). TriP, triphosphorylated RNA; DiP, diphosphorylated RNA; MonoP, monophosphorylated RNA.

    Techniques Used: Ligation, Electrophoresis, Northern Blot

    9) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.284992

    Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and
    Figure Legend Snippet: Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and

    Techniques Used:

    Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .
    Figure Legend Snippet: Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .

    Techniques Used: Diffusion-based Assay, Binding Assay

    Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (
    Figure Legend Snippet: Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (

    Techniques Used:

    10) Product Images from "Enhancer RNA-driven looping enhances the transcription of the long noncoding RNA DHRS4-AS1, a controller of the DHRS4 gene cluster"

    Article Title: Enhancer RNA-driven looping enhances the transcription of the long noncoding RNA DHRS4-AS1, a controller of the DHRS4 gene cluster

    Journal: Scientific Reports

    doi: 10.1038/srep20961

    AS1 enhancer selectively interacts with DHRS4-AS1 promoter. Cells were crosslinked with 1% formaldehyde, and then the reaction was stopped by the addition of glycine. Restriction enzymes Hind III or Eco RI were used to digest the crosslinked chromatin. Then ligation was performed by incubation with T4 DNA ligase. Purified ligation products were determined by qPCR. ( A ) The relative crosslinking frequencies between the anchor region (the AS1 enhancer) and distal fragments (F1~F5) were measured by qPCR and normalized to the control region (fragment F1). Error bars indicate the mean ± SEM of three experiments. P values were determined by Student’s unpaired two-tailed t test. ( B , C ) Spatial interactions between the AS1 enhancer and homologous promoter regions of putative DHRS4L2 and DHRS4L1 NATs were determined by 3C array. Crosslinked chromatin was then digested with Hind III ( B ) or Eco RI ( C ), followed by ligation. 3C samples from Hind III- ( B ) or Eco RI- ( C ) digested crosslinked chromatin without ligation and non-crosslinked genomic DNA with or without ligation were used as negative controls. To ensure the various primer pairs all worked as intended, we performed a random ligation control where PCR was performed to amplify DNA from BAC clones. The primers used here effectively amplified the control templates that contained all the ligation products. Notably, the putative promoter sequences used in this analysis contain all the potential promoters predicted from PROSCAN (see Methods) at the putative homologous NATs of DHRS4L2 and DHRS4L1 . The PCR products from 3C samples, not cut with any restriction enzyme, were used as the loading control.
    Figure Legend Snippet: AS1 enhancer selectively interacts with DHRS4-AS1 promoter. Cells were crosslinked with 1% formaldehyde, and then the reaction was stopped by the addition of glycine. Restriction enzymes Hind III or Eco RI were used to digest the crosslinked chromatin. Then ligation was performed by incubation with T4 DNA ligase. Purified ligation products were determined by qPCR. ( A ) The relative crosslinking frequencies between the anchor region (the AS1 enhancer) and distal fragments (F1~F5) were measured by qPCR and normalized to the control region (fragment F1). Error bars indicate the mean ± SEM of three experiments. P values were determined by Student’s unpaired two-tailed t test. ( B , C ) Spatial interactions between the AS1 enhancer and homologous promoter regions of putative DHRS4L2 and DHRS4L1 NATs were determined by 3C array. Crosslinked chromatin was then digested with Hind III ( B ) or Eco RI ( C ), followed by ligation. 3C samples from Hind III- ( B ) or Eco RI- ( C ) digested crosslinked chromatin without ligation and non-crosslinked genomic DNA with or without ligation were used as negative controls. To ensure the various primer pairs all worked as intended, we performed a random ligation control where PCR was performed to amplify DNA from BAC clones. The primers used here effectively amplified the control templates that contained all the ligation products. Notably, the putative promoter sequences used in this analysis contain all the potential promoters predicted from PROSCAN (see Methods) at the putative homologous NATs of DHRS4L2 and DHRS4L1 . The PCR products from 3C samples, not cut with any restriction enzyme, were used as the loading control.

    Techniques Used: Ligation, Incubation, Purification, Real-time Polymerase Chain Reaction, Two Tailed Test, Polymerase Chain Reaction, BAC Assay, Clone Assay, Amplification

    AS1eRNA enhances DHRS4-AS1 transcription by mediating DNA looping between the DHRS4-AS1 promoter and its enhancer. ( A ) AS1eRNA is stable following treatment with actinomycin D. Cells were treated with 5 μg/ml actinomycin D, using c-MYC as the positive control in HepG2 and HL7702 cells. ( B,C ) After siAS1eRNA treatment, qPCR analysis of AS1eRNA and DHRS4-AS1 levels was performed at 48 h. The analysis of RNA levels used β-actin as the internal control. The siRNA-control was a scrambled sequence with no homology to any known gene. ( D ) The relative crosslinking frequencies of the AS1 enhancer/ DHRS4-AS1 promoter were determined by qPCR. We first normalized the bias of the PCR efficiency among each primer set we used. This was performed by measuring the corresponding amplifications from a bacterial artificial chromosome, which was digested with Hind III and ligated with T4 DNA ligase. For correcting the differences of crosslinking and digestion efficiencies between samples, ERCC3 “control interaction frequencies” were used as internal control (see Methods). The relative crosslinking frequencies of F1~F5 was determined the fold change relative to the control region (F1). P values were determined by Student’s unpaired two-tailed t test. Error bars represent the mean ± SEM of three independent experiments. * P
    Figure Legend Snippet: AS1eRNA enhances DHRS4-AS1 transcription by mediating DNA looping between the DHRS4-AS1 promoter and its enhancer. ( A ) AS1eRNA is stable following treatment with actinomycin D. Cells were treated with 5 μg/ml actinomycin D, using c-MYC as the positive control in HepG2 and HL7702 cells. ( B,C ) After siAS1eRNA treatment, qPCR analysis of AS1eRNA and DHRS4-AS1 levels was performed at 48 h. The analysis of RNA levels used β-actin as the internal control. The siRNA-control was a scrambled sequence with no homology to any known gene. ( D ) The relative crosslinking frequencies of the AS1 enhancer/ DHRS4-AS1 promoter were determined by qPCR. We first normalized the bias of the PCR efficiency among each primer set we used. This was performed by measuring the corresponding amplifications from a bacterial artificial chromosome, which was digested with Hind III and ligated with T4 DNA ligase. For correcting the differences of crosslinking and digestion efficiencies between samples, ERCC3 “control interaction frequencies” were used as internal control (see Methods). The relative crosslinking frequencies of F1~F5 was determined the fold change relative to the control region (F1). P values were determined by Student’s unpaired two-tailed t test. Error bars represent the mean ± SEM of three independent experiments. * P

    Techniques Used: Positive Control, Real-time Polymerase Chain Reaction, Sequencing, Polymerase Chain Reaction, Two Tailed Test

    11) Product Images from "An optogenetic tool to recruit individual PKC isozymes to the cell surface and promote specific phosphorylation of membrane proteins"

    Article Title: An optogenetic tool to recruit individual PKC isozymes to the cell surface and promote specific phosphorylation of membrane proteins

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2022.101893

    Schematic drawing depicting constructs involved in optogenetic activation of PKCε-CAT. A , schematic of the subdomains within the PKCε isozyme. B , digestion of parent constructs mCherry-CRY2-5Ptase OCRL and mPKCε-CAT-HA-pXOOM; ligation of fragments with T4 DNA ligase. C , blue light induced recruitment of mCherry-CRY2–mPKCε-CAT-HA to the cell surface and the subsequent phosphorylation of membrane protein substrates by PKCε. CAT, catalytic domain; CRY2, cryptochrome-2; HA, hemagglutinin.
    Figure Legend Snippet: Schematic drawing depicting constructs involved in optogenetic activation of PKCε-CAT. A , schematic of the subdomains within the PKCε isozyme. B , digestion of parent constructs mCherry-CRY2-5Ptase OCRL and mPKCε-CAT-HA-pXOOM; ligation of fragments with T4 DNA ligase. C , blue light induced recruitment of mCherry-CRY2–mPKCε-CAT-HA to the cell surface and the subsequent phosphorylation of membrane protein substrates by PKCε. CAT, catalytic domain; CRY2, cryptochrome-2; HA, hemagglutinin.

    Techniques Used: Construct, Activation Assay, Ligation

    12) Product Images from "Genetic diversity and mother-child overlap of the gut associated microbiota determined by reduced genome sequencing"

    Article Title: Genetic diversity and mother-child overlap of the gut associated microbiota determined by reduced genome sequencing

    Journal: bioRxiv

    doi: 10.1101/191445

    Evaluation of (A) the uniqueness of the reduced metagenome fragments and (B) the quantitative properties. The true concentrations are based amount of DNA added for the different species.
    Figure Legend Snippet: Evaluation of (A) the uniqueness of the reduced metagenome fragments and (B) the quantitative properties. The true concentrations are based amount of DNA added for the different species.

    Techniques Used:

    13) Product Images from "The Putative Endonuclease Activity of MutL Is Required for the Segmental Gene Conversion Events That Drive Antigenic Variation of the Lyme Disease Spirochete"

    Article Title: The Putative Endonuclease Activity of MutL Is Required for the Segmental Gene Conversion Events That Drive Antigenic Variation of the Lyme Disease Spirochete

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2022.888494

    Schematic of plasmid construction and introduction of point mutations into B. burgdorferi chromosomal mutL . (A) To generate the E. coli plasmids carrying point mutations in mutL we designed a two-insert strategy where two mutL double stranded fragments carrying complementary base pair changes (represented as an X) were ligated with each other and to pJET1.2 using the HiFi DNA Assembly mix from NEB. This assembly mix contains a 5′-3′ exonuclease that generates single stranded 3′ overhangs. These overhangs are able to anneal for a high-fidelity polymerase to extend the overhangs and fill in the gaps; finally, a DNA ligase seals the nicks. (B) The pJET plasmids containing the mutated versions of mutL were then digested with Xho I and Nco I, and (C) the mutated mutL inserts were then cloned into pOK12, (D) generating the plasmids pMC140, pMC141, pMC142, and pMC143 (see Supplementary Table 3 ). We then inserted the gentamicin resistance cassette with its own promoter (black arrow) downstream of mutL at the Nco I site. (E) Finally, we cloned the bb0212 gene downstream of the gent cassette to maintain the natural gene order in the B. burgdorferi chromosome. (F) Plasmids pMC148, pMC149, pMC150, and pMC151 were used to transform B. burgdorferi B31 wild-type clone 5A4 [20] and (G) GentR-KanS transformants were further analyzed for double recombination events.
    Figure Legend Snippet: Schematic of plasmid construction and introduction of point mutations into B. burgdorferi chromosomal mutL . (A) To generate the E. coli plasmids carrying point mutations in mutL we designed a two-insert strategy where two mutL double stranded fragments carrying complementary base pair changes (represented as an X) were ligated with each other and to pJET1.2 using the HiFi DNA Assembly mix from NEB. This assembly mix contains a 5′-3′ exonuclease that generates single stranded 3′ overhangs. These overhangs are able to anneal for a high-fidelity polymerase to extend the overhangs and fill in the gaps; finally, a DNA ligase seals the nicks. (B) The pJET plasmids containing the mutated versions of mutL were then digested with Xho I and Nco I, and (C) the mutated mutL inserts were then cloned into pOK12, (D) generating the plasmids pMC140, pMC141, pMC142, and pMC143 (see Supplementary Table 3 ). We then inserted the gentamicin resistance cassette with its own promoter (black arrow) downstream of mutL at the Nco I site. (E) Finally, we cloned the bb0212 gene downstream of the gent cassette to maintain the natural gene order in the B. burgdorferi chromosome. (F) Plasmids pMC148, pMC149, pMC150, and pMC151 were used to transform B. burgdorferi B31 wild-type clone 5A4 [20] and (G) GentR-KanS transformants were further analyzed for double recombination events.

    Techniques Used: Plasmid Preparation, Clone Assay

    14) Product Images from "Synapsis of Recombination Signal Sequences Located in cis and DNA Underwinding in V(D)J Recombination"

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

    Journal:

    doi: 10.1128/MCB.24.19.8727-8744.2004

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

    Techniques Used: Ligation

    15) Product Images from "A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL"

    Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky708

    RNase H1 processing coupled to POLγ dependent DNA synthesis does not produce ligatable nicks. ( A ) Schematic of the coupled nuclease gap-filling ligation assay performed on a gapped OriL substrate (a). The upstream oligonucleotide was radioactively labelled at the 5′-end. The possible products are illustrated (b–d). (–) Coupled nuclease gap-filling ligation assay as shown in A. POLγ filled the gap (lane 2, marked b) and had limited strand displacement activity (SD). Note though that POLγ completely displaces the downstream oligonucleotide in a small fraction of templates (80 nt band, lanes 3–6,), RNase H1 cleaved the RNA in the substrate (lane 3), enabling further gap-filling (marked c). Only very low levels of ligated products were formed in the presence of 80–320 fmol DNA ligase III (lanes 4–6). A prominent 80 nt ligated product was formed with T4 DNA ligase (lane 7, marked d). The letters a-d correspond to the illustrations in panel A. ( C ) Ligation assay on a nicked substrate containing RNA tracts of varying length downstream of the nick in the presence of 80–320 fmol DNA ligase III. DNA ligase III discriminates against nicked substrates that contain increasing stretches of ribonucleotides. Two, but not five or more ribonucleotides, can be ligated. ( D ) As in C, except performed with T4 ligase (1–8 U). T4 ligase can ligate five but not 10 ribonucleotides. ( E ) T4 ligase-mediated ligation is abolished in the presence of the mutant RNase H1 proteins. The letters a-d correspond to the illustrations in panel A.
    Figure Legend Snippet: RNase H1 processing coupled to POLγ dependent DNA synthesis does not produce ligatable nicks. ( A ) Schematic of the coupled nuclease gap-filling ligation assay performed on a gapped OriL substrate (a). The upstream oligonucleotide was radioactively labelled at the 5′-end. The possible products are illustrated (b–d). (–) Coupled nuclease gap-filling ligation assay as shown in A. POLγ filled the gap (lane 2, marked b) and had limited strand displacement activity (SD). Note though that POLγ completely displaces the downstream oligonucleotide in a small fraction of templates (80 nt band, lanes 3–6,), RNase H1 cleaved the RNA in the substrate (lane 3), enabling further gap-filling (marked c). Only very low levels of ligated products were formed in the presence of 80–320 fmol DNA ligase III (lanes 4–6). A prominent 80 nt ligated product was formed with T4 DNA ligase (lane 7, marked d). The letters a-d correspond to the illustrations in panel A. ( C ) Ligation assay on a nicked substrate containing RNA tracts of varying length downstream of the nick in the presence of 80–320 fmol DNA ligase III. DNA ligase III discriminates against nicked substrates that contain increasing stretches of ribonucleotides. Two, but not five or more ribonucleotides, can be ligated. ( D ) As in C, except performed with T4 ligase (1–8 U). T4 ligase can ligate five but not 10 ribonucleotides. ( E ) T4 ligase-mediated ligation is abolished in the presence of the mutant RNase H1 proteins. The letters a-d correspond to the illustrations in panel A.

    Techniques Used: DNA Synthesis, Ligation, Activity Assay, Mutagenesis

    16) Product Images from "Efficient assembly of very short oligonucleotides using T4 DNA Ligase"

    Article Title: Efficient assembly of very short oligonucleotides using T4 DNA Ligase

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-3-291

    Enhancement of T4 DNA ligase activity by supplemental oligonucleotides. (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first oligonucleotide-dsDNA duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.
    Figure Legend Snippet: Enhancement of T4 DNA ligase activity by supplemental oligonucleotides. (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first oligonucleotide-dsDNA duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.

    Techniques Used: Activity Assay, Ligation

    Evaluation of minimal oligonucleotide substrate requirements for T4 DNA ligase. (a) Schematic diagram of an immobilized DNA strand used in ligation assays and DNA construction. M-270 Dynabeads (Invitrogen) are attached through a streptavidin-biotin linkage to the 5' end of a double stranded DNA. The free end is designed with a variable 5' overhang, complementary to labeled oligonucleotides used in ligation. An additional BbsI restriction site and a forward primer site are included in the case of DNA construction. (b) Increasing concentrations of 5'-phosphorylated, 3'-fluorescently labeled oligonucleotide are ligated to 5 pmoles of immobilized dsDNA with a complementary overhang. Reactions were performed for one hour at 16°C and washed with TE to remove unligated substrate. Successful ligation kinetics are observed at the 5-bp duplex length (▲), but no significant ligation occurs at lengths of 4-bp (■) or 3-bp (◆).
    Figure Legend Snippet: Evaluation of minimal oligonucleotide substrate requirements for T4 DNA ligase. (a) Schematic diagram of an immobilized DNA strand used in ligation assays and DNA construction. M-270 Dynabeads (Invitrogen) are attached through a streptavidin-biotin linkage to the 5' end of a double stranded DNA. The free end is designed with a variable 5' overhang, complementary to labeled oligonucleotides used in ligation. An additional BbsI restriction site and a forward primer site are included in the case of DNA construction. (b) Increasing concentrations of 5'-phosphorylated, 3'-fluorescently labeled oligonucleotide are ligated to 5 pmoles of immobilized dsDNA with a complementary overhang. Reactions were performed for one hour at 16°C and washed with TE to remove unligated substrate. Successful ligation kinetics are observed at the 5-bp duplex length (▲), but no significant ligation occurs at lengths of 4-bp (■) or 3-bp (◆).

    Techniques Used: Ligation, Labeling

    17) Product Images from "Optimizing the design of protein nanoparticles as carriers for vaccine applications"

    Article Title: Optimizing the design of protein nanoparticles as carriers for vaccine applications

    Journal: Nanomedicine : nanotechnology, biology, and medicine

    doi: 10.1016/j.nano.2015.05.003

    (a) PDB search for protein structures with inter-helical angles similar to the mode of the nanoparticle followed by superposition of the helices of the different pdb-structures (blue and red) onto the pentamer (green) and trimer (blue) helices of the monomer of the peptide nanoparticle. The residues with angles similar to the angle between the helices of the pentamer and the trimer in the peptide nanoparticle were selected (gray region). ( b) Schematics of the molecular biology strategy for inserting the new linker region: the original construct with pentameric (green) and trimeric (blue) helices is double digested with restriction enzymes ApaI and XhoI. ( c) Linker oligonucleotides selected from panel a are ligated into double digested vector (panel b ) using T4 DNA ligase generating new plasmid which codes for the genetically modified single polypeptide chain.
    Figure Legend Snippet: (a) PDB search for protein structures with inter-helical angles similar to the mode of the nanoparticle followed by superposition of the helices of the different pdb-structures (blue and red) onto the pentamer (green) and trimer (blue) helices of the monomer of the peptide nanoparticle. The residues with angles similar to the angle between the helices of the pentamer and the trimer in the peptide nanoparticle were selected (gray region). ( b) Schematics of the molecular biology strategy for inserting the new linker region: the original construct with pentameric (green) and trimeric (blue) helices is double digested with restriction enzymes ApaI and XhoI. ( c) Linker oligonucleotides selected from panel a are ligated into double digested vector (panel b ) using T4 DNA ligase generating new plasmid which codes for the genetically modified single polypeptide chain.

    Techniques Used: Construct, Plasmid Preparation, Genetically Modified

    18) Product Images from "Probing transient protein-mediated DNA linkages using nanoconfinement"

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

    Journal: Biomicrofluidics

    doi: 10.1063/1.4882775

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

    Techniques Used: Binding Assay

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

    Techniques Used:

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

    Techniques Used:

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

    Techniques Used:

    19) Product Images from "Analytical biochemistry of DNA-protein assemblies from crude cell extracts"

    Article Title: Analytical biochemistry of DNA-protein assemblies from crude cell extracts

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm490

    Oligonucleotide ligation by DNA ends-binding protein complexes. After washes, the DNA–protein assemblies were incubated with ATP to allow the ligation of the TetO sequence. In lane 3, the ligation was performed on the beads in presence of ATP then the nucleic sequence was released after irradiation of the chromatographic slurry. In lane 4, the DNA–protein assemblies were recovered after the irradiation step then incubated with ATP. The oligonucleotides were resolved on an 8% non-denaturing polyacrylamide gel and revealed by Southern blot with the radiolabeled non-photocleavable strand of the PCB-TetO duplex. Lane 1 shows the intact TetO oligonucleotide and lane 2 the oligonucleotide ligated by the T4 DNA ligase protein.
    Figure Legend Snippet: Oligonucleotide ligation by DNA ends-binding protein complexes. After washes, the DNA–protein assemblies were incubated with ATP to allow the ligation of the TetO sequence. In lane 3, the ligation was performed on the beads in presence of ATP then the nucleic sequence was released after irradiation of the chromatographic slurry. In lane 4, the DNA–protein assemblies were recovered after the irradiation step then incubated with ATP. The oligonucleotides were resolved on an 8% non-denaturing polyacrylamide gel and revealed by Southern blot with the radiolabeled non-photocleavable strand of the PCB-TetO duplex. Lane 1 shows the intact TetO oligonucleotide and lane 2 the oligonucleotide ligated by the T4 DNA ligase protein.

    Techniques Used: Ligation, Binding Assay, Incubation, Sequencing, Irradiation, Southern Blot

    20) Product Images from "DNA supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia coli"

    Article Title: DNA supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia coli

    Journal: Scientific Reports

    doi: 10.1038/srep19243

    One molecule of LacI tetramer divided a supercoiled DNA molecule plasmid pCB126 into two independent topological domains. ( a ) Plasmid pCB126 carrying two lac O1 operators in two different locations was constructed as detailed in Methods. ( b ) The nicking enzyme Nt.BbvCI was able to rapidly digest pCB126. Time course of enzyme digestion of pCB126 using 16 units of Nt.BbvCI in 1 × NEBuffer 4 at 37 °C. Lane 1 contained the undigested scDNA. ( c ) Time course of DNA supercoiling diffusion in the presence of LacI. The DNA-nicking assays were performed as described under Methods. Each reaction mixture (320 μL) contained 0.156 nM of pCB126, 2.5 nM of LacI, and 16 units of Nt.BbvCI. The reactions were incubated at 37 °C for the time indicated. Then a large excess of a double-stranded oligonucleotide contain an Nt.BbvCI recognition site was added to the reaction mixture to inhibit the restriction enzyme activities. The nicked DNA templates were ligated by T4 DNA ligase in the presence of 1 mM of ATP at 37 °C for 5 min and the reactions were terminated by phenol extraction. The DNA molecules were isolated and subjected to agarose gel electrophoresis. ( d ) Quantification analysis of the time course. The percentage of supercoiled DNA was plotted against the reaction time. The curve was generated by fitting the data to a 1st-order rate equation to yield a rate constant of 0.016 sec −1 and a t 1/2 of 52 sec.
    Figure Legend Snippet: One molecule of LacI tetramer divided a supercoiled DNA molecule plasmid pCB126 into two independent topological domains. ( a ) Plasmid pCB126 carrying two lac O1 operators in two different locations was constructed as detailed in Methods. ( b ) The nicking enzyme Nt.BbvCI was able to rapidly digest pCB126. Time course of enzyme digestion of pCB126 using 16 units of Nt.BbvCI in 1 × NEBuffer 4 at 37 °C. Lane 1 contained the undigested scDNA. ( c ) Time course of DNA supercoiling diffusion in the presence of LacI. The DNA-nicking assays were performed as described under Methods. Each reaction mixture (320 μL) contained 0.156 nM of pCB126, 2.5 nM of LacI, and 16 units of Nt.BbvCI. The reactions were incubated at 37 °C for the time indicated. Then a large excess of a double-stranded oligonucleotide contain an Nt.BbvCI recognition site was added to the reaction mixture to inhibit the restriction enzyme activities. The nicked DNA templates were ligated by T4 DNA ligase in the presence of 1 mM of ATP at 37 °C for 5 min and the reactions were terminated by phenol extraction. The DNA molecules were isolated and subjected to agarose gel electrophoresis. ( d ) Quantification analysis of the time course. The percentage of supercoiled DNA was plotted against the reaction time. The curve was generated by fitting the data to a 1st-order rate equation to yield a rate constant of 0.016 sec −1 and a t 1/2 of 52 sec.

    Techniques Used: Plasmid Preparation, Construct, Diffusion-based Assay, Incubation, Isolation, Agarose Gel Electrophoresis, Generated, Size-exclusion Chromatography

    21) Product Images from "The Sole DNA Ligase in Entamoeba histolytica Is a High-Fidelity DNA Ligase Involved in DNA Damage Repair"

    Article Title: The Sole DNA Ligase in Entamoeba histolytica Is a High-Fidelity DNA Ligase Involved in DNA Damage Repair

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2018.00214

    EhDNALigI activity on substrates with oxidative stress damage. (A) Schematic representation of the assayed substrate to measure DNA ligation at damaged substrates. The “X” indicates the location of three oxidative damages: 8-oxoG, thymine glycol and abasic site. In all cases, the damage is located at the template strand, while the complementary strand may have an adenine or cytosine, located at the 3′-OH (acceptor) (A' and B') or 5′-PO 4 (donor) (C' and D'). (B) Ligation reaction with 8-oxoG template. Enzymatic activity against damaged substrates with different combinations as shown in (A) using EhDNAligI (lanes 1–4) or T4 DNA ligase (lanes 5–8). (C) Ligation reaction with thymine glycol template. Enzymatic activity against damaged substrates with different combinations as shown in (A) using EhDNAligI (lanes 1–4) or T4 DNA ligase (lanes 5–8). (D) Ligation reaction with abasic site. Enzymatic activity against damaged substrates with different combinations as shown in (A) using EhDNAligI (lanes 1–4) or T4 DNA ligase (lanes 5–8). pDNA indicates the phosphorylated substrate DNA, while AppDNA indicates adenylated substrate by a DNA ligase. The substrate and the ligation product are labeled with arrows.
    Figure Legend Snippet: EhDNALigI activity on substrates with oxidative stress damage. (A) Schematic representation of the assayed substrate to measure DNA ligation at damaged substrates. The “X” indicates the location of three oxidative damages: 8-oxoG, thymine glycol and abasic site. In all cases, the damage is located at the template strand, while the complementary strand may have an adenine or cytosine, located at the 3′-OH (acceptor) (A' and B') or 5′-PO 4 (donor) (C' and D'). (B) Ligation reaction with 8-oxoG template. Enzymatic activity against damaged substrates with different combinations as shown in (A) using EhDNAligI (lanes 1–4) or T4 DNA ligase (lanes 5–8). (C) Ligation reaction with thymine glycol template. Enzymatic activity against damaged substrates with different combinations as shown in (A) using EhDNAligI (lanes 1–4) or T4 DNA ligase (lanes 5–8). (D) Ligation reaction with abasic site. Enzymatic activity against damaged substrates with different combinations as shown in (A) using EhDNAligI (lanes 1–4) or T4 DNA ligase (lanes 5–8). pDNA indicates the phosphorylated substrate DNA, while AppDNA indicates adenylated substrate by a DNA ligase. The substrate and the ligation product are labeled with arrows.

    Techniques Used: Activity Assay, DNA Ligation, Ligation, Labeling

    22) Product Images from "Purely enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR"

    Article Title: Purely enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR

    Journal: bioRxiv

    doi: 10.1101/2022.02.16.480708

    Scheme illustrating the workflow of A31-labeling of target RNA (46 nt). A: Separate production of two RNA fragments. The 3’-RNA fragment (orange) starts with the labeled nucleotide A31, which is supplied as 13 C/ 15 N-AMP into the in vitro transcription reaction. B: Ligation of unlabeled 5’-RNA (purple) and A1-labeled 3’-RNA fragment (orange) using T4 DNA ligase to produce the A31-labeled full-length RNA construct. C: Final A31-labeled RNA construct comprised of unlabeled 3’-RNA (purple) and A1-labeled (orange).
    Figure Legend Snippet: Scheme illustrating the workflow of A31-labeling of target RNA (46 nt). A: Separate production of two RNA fragments. The 3’-RNA fragment (orange) starts with the labeled nucleotide A31, which is supplied as 13 C/ 15 N-AMP into the in vitro transcription reaction. B: Ligation of unlabeled 5’-RNA (purple) and A1-labeled 3’-RNA fragment (orange) using T4 DNA ligase to produce the A31-labeled full-length RNA construct. C: Final A31-labeled RNA construct comprised of unlabeled 3’-RNA (purple) and A1-labeled (orange).

    Techniques Used: Labeling, In Vitro, Ligation, Construct

    Confirmation of A31-label incorporation into the 46 nt full-length RNA. A: Denaturing PAGE of ligation reaction. Size reference nt* refers to a DNA oligo (ligation splint). Lane 1: Purified 5’-RNA fragment (purple box); Lane 2: Purified 3’-RNA fragment (orange box); Lane 3: Negative control of ligation reaction without T4 DNA ligase. Lane 4: Ligation reaction after 48 h. Lane 5: HPLC-purified full-length RNA. B: 1 H, 13 C-HSQC of A1-labeled 3’-RNA fragment (16 nt) showing the two expected signals for C8 and C2 of A1. C: 1 H, 13 C-HSQC spectra of the aromatic region of A31-labeled full-length RNA (red) and uniformly A/U-labeled full-length RNA (blue). For the A31-labeled RNA, two dominant set of C8/C2 signals appear alongside a weaker set of signals (A31C2* and A31C8*). The zoom shows that A31C2 does indeed overlay directly with a signal of the A/U-labeled RNA, which is partially overlapped with another signal. Furthermore, clear differences between 2B and 2C indicate proper incorporation of the isotope-labeled nucleotide into the RNA. A secondary structure prediction( 25 ) shows which nucleotides are expected to give HSQC signals for A31-labeled RNA (red) or A/U-labeled RNA (blue).
    Figure Legend Snippet: Confirmation of A31-label incorporation into the 46 nt full-length RNA. A: Denaturing PAGE of ligation reaction. Size reference nt* refers to a DNA oligo (ligation splint). Lane 1: Purified 5’-RNA fragment (purple box); Lane 2: Purified 3’-RNA fragment (orange box); Lane 3: Negative control of ligation reaction without T4 DNA ligase. Lane 4: Ligation reaction after 48 h. Lane 5: HPLC-purified full-length RNA. B: 1 H, 13 C-HSQC of A1-labeled 3’-RNA fragment (16 nt) showing the two expected signals for C8 and C2 of A1. C: 1 H, 13 C-HSQC spectra of the aromatic region of A31-labeled full-length RNA (red) and uniformly A/U-labeled full-length RNA (blue). For the A31-labeled RNA, two dominant set of C8/C2 signals appear alongside a weaker set of signals (A31C2* and A31C8*). The zoom shows that A31C2 does indeed overlay directly with a signal of the A/U-labeled RNA, which is partially overlapped with another signal. Furthermore, clear differences between 2B and 2C indicate proper incorporation of the isotope-labeled nucleotide into the RNA. A secondary structure prediction( 25 ) shows which nucleotides are expected to give HSQC signals for A31-labeled RNA (red) or A/U-labeled RNA (blue).

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Purification, Negative Control, High Performance Liquid Chromatography, Labeling

    23) Product Images from "Purely enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR"

    Article Title: Purely enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR

    Journal: bioRxiv

    doi: 10.1101/2022.02.16.480708

    Scheme illustrating the workflow of A31-labeling of target RNA (46 nt). A: Separate production of two RNA fragments. The 3’-RNA fragment (orange) starts with the labeled nucleotide A31, which is supplied as 13 C/ 15 N-AMP into the in vitro transcription reaction. B: Ligation of unlabeled 5’-RNA (purple) and A1-labeled 3’-RNA fragment (orange) using T4 DNA ligase to produce the A31-labeled full-length RNA construct. C: Final A31-labeled RNA construct comprised of unlabeled 3’-RNA (purple) and A1-labeled (orange).
    Figure Legend Snippet: Scheme illustrating the workflow of A31-labeling of target RNA (46 nt). A: Separate production of two RNA fragments. The 3’-RNA fragment (orange) starts with the labeled nucleotide A31, which is supplied as 13 C/ 15 N-AMP into the in vitro transcription reaction. B: Ligation of unlabeled 5’-RNA (purple) and A1-labeled 3’-RNA fragment (orange) using T4 DNA ligase to produce the A31-labeled full-length RNA construct. C: Final A31-labeled RNA construct comprised of unlabeled 3’-RNA (purple) and A1-labeled (orange).

    Techniques Used: Labeling, In Vitro, Ligation, Construct

    Confirmation of A31-label incorporation into the 46 nt full-length RNA. A: Denaturing PAGE of ligation reaction. Size reference nt* refers to a DNA oligo (ligation splint). Lane 1: Purified 5’-RNA fragment (purple box); Lane 2: Purified 3’-RNA fragment (orange box); Lane 3: Negative control of ligation reaction without T4 DNA ligase. Lane 4: Ligation reaction after 48 h. Lane 5: HPLC-purified full-length RNA. B: 1 H, 13 C-HSQC of A1-labeled 3’-RNA fragment (16 nt) showing the two expected signals for C8 and C2 of A1. C: 1 H, 13 C-HSQC spectra of the aromatic region of A31-labeled full-length RNA (red) and uniformly A/U-labeled full-length RNA (blue). For the A31-labeled RNA, two dominant set of C8/C2 signals appear alongside a weaker set of signals (A31C2* and A31C8*). The zoom shows that A31C2 does indeed overlay directly with a signal of the A/U-labeled RNA, which is partially overlapped with another signal. Furthermore, clear differences between 2B and 2C indicate proper incorporation of the isotope-labeled nucleotide into the RNA. A secondary structure prediction( 25 ) shows which nucleotides are expected to give HSQC signals for A31-labeled RNA (red) or A/U-labeled RNA (blue).
    Figure Legend Snippet: Confirmation of A31-label incorporation into the 46 nt full-length RNA. A: Denaturing PAGE of ligation reaction. Size reference nt* refers to a DNA oligo (ligation splint). Lane 1: Purified 5’-RNA fragment (purple box); Lane 2: Purified 3’-RNA fragment (orange box); Lane 3: Negative control of ligation reaction without T4 DNA ligase. Lane 4: Ligation reaction after 48 h. Lane 5: HPLC-purified full-length RNA. B: 1 H, 13 C-HSQC of A1-labeled 3’-RNA fragment (16 nt) showing the two expected signals for C8 and C2 of A1. C: 1 H, 13 C-HSQC spectra of the aromatic region of A31-labeled full-length RNA (red) and uniformly A/U-labeled full-length RNA (blue). For the A31-labeled RNA, two dominant set of C8/C2 signals appear alongside a weaker set of signals (A31C2* and A31C8*). The zoom shows that A31C2 does indeed overlay directly with a signal of the A/U-labeled RNA, which is partially overlapped with another signal. Furthermore, clear differences between 2B and 2C indicate proper incorporation of the isotope-labeled nucleotide into the RNA. A secondary structure prediction( 25 ) shows which nucleotides are expected to give HSQC signals for A31-labeled RNA (red) or A/U-labeled RNA (blue).

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Purification, Negative Control, High Performance Liquid Chromatography, Labeling

    24) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.284992

    Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and
    Figure Legend Snippet: Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and

    Techniques Used:

    Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .
    Figure Legend Snippet: Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .

    Techniques Used: Diffusion-based Assay, Binding Assay

    Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (
    Figure Legend Snippet: Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (

    Techniques Used:

    25) Product Images from "Mechanism and resistance for antimycobacterial activity of a fluoroquinophenoxazine compound"

    Article Title: Mechanism and resistance for antimycobacterial activity of a fluoroquinophenoxazine compound

    Journal: bioRxiv

    doi: 10.1101/464990

    Products of DNA ligation in the presence of FP-11g. DNA ligation of nicked pAB1 plasmid DNA by T4 DNA ligase in the presence of indicated concentrations of FP-11g were performed as described under Methods.
    Figure Legend Snippet: Products of DNA ligation in the presence of FP-11g. DNA ligation of nicked pAB1 plasmid DNA by T4 DNA ligase in the presence of indicated concentrations of FP-11g were performed as described under Methods.

    Techniques Used: DNA Ligation, Plasmid Preparation

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    New England Biolabs t4 dna ligase
    FEN1 completes primer removal and L-strand maturation. ( A ) Schematic of template used in panel B. The possible products are illustrated (b–d). ( B ) Coupled nuclease gap-filling ligation assay was performed as in Figure 3 but in the presence of the indicated nucleases. MGME1 (250 fmol) was added in lanes 4 and 9; FEN1 (35 fmol) was added in lanes 5 and 10. The samples in lanes 6–10 contained 300 fmol DNA ligase III. Conversion of the nicked product (c) to a ligated 80 nt product (d) was stimulated when FEN1 was added together with RNase H1. ( C ) Schematic of template used in panel D. The downstream chimeric oligonucleotide was labelled at the 3′-end. The possible products are illustrated (b–d). ( D ) Coupled nuclease gap-filling ligation assay was performed in the presence of RNase H1 alone or together with FEN1 (35 fmol) as indicated. Lanes 2–4 contained only RNase H1 with or without FEN1 to monitor nuclease activity. RNase H1 cleaved the downstream oligonucleotide leaving 1–3 unprocessed ribonucleotides. FEN1 cleaved the remaining ribonucleotides, resulting in a shorter product. POLγ and DNA ligase (ligase III or <t>T4</t> DNA ligase) were added in lanes 5–10. Ligase III showed ligation only when both RNase H1 and FEN1 were added. However, T4 DNA ligase could ligate without FEN1. Lanes 12–14 are ligation controls using a 3′-end labelled DNA-only oligonucleotide with a 5′-end phosphate. Note that the short band (
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 dna ligase reaction buffer
    YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with <t>T4</t> DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .
    T4 Dna Ligase Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FEN1 completes primer removal and L-strand maturation. ( A ) Schematic of template used in panel B. The possible products are illustrated (b–d). ( B ) Coupled nuclease gap-filling ligation assay was performed as in Figure 3 but in the presence of the indicated nucleases. MGME1 (250 fmol) was added in lanes 4 and 9; FEN1 (35 fmol) was added in lanes 5 and 10. The samples in lanes 6–10 contained 300 fmol DNA ligase III. Conversion of the nicked product (c) to a ligated 80 nt product (d) was stimulated when FEN1 was added together with RNase H1. ( C ) Schematic of template used in panel D. The downstream chimeric oligonucleotide was labelled at the 3′-end. The possible products are illustrated (b–d). ( D ) Coupled nuclease gap-filling ligation assay was performed in the presence of RNase H1 alone or together with FEN1 (35 fmol) as indicated. Lanes 2–4 contained only RNase H1 with or without FEN1 to monitor nuclease activity. RNase H1 cleaved the downstream oligonucleotide leaving 1–3 unprocessed ribonucleotides. FEN1 cleaved the remaining ribonucleotides, resulting in a shorter product. POLγ and DNA ligase (ligase III or T4 DNA ligase) were added in lanes 5–10. Ligase III showed ligation only when both RNase H1 and FEN1 were added. However, T4 DNA ligase could ligate without FEN1. Lanes 12–14 are ligation controls using a 3′-end labelled DNA-only oligonucleotide with a 5′-end phosphate. Note that the short band (

    Journal: Nucleic Acids Research

    Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL

    doi: 10.1093/nar/gky708

    Figure Lengend Snippet: FEN1 completes primer removal and L-strand maturation. ( A ) Schematic of template used in panel B. The possible products are illustrated (b–d). ( B ) Coupled nuclease gap-filling ligation assay was performed as in Figure 3 but in the presence of the indicated nucleases. MGME1 (250 fmol) was added in lanes 4 and 9; FEN1 (35 fmol) was added in lanes 5 and 10. The samples in lanes 6–10 contained 300 fmol DNA ligase III. Conversion of the nicked product (c) to a ligated 80 nt product (d) was stimulated when FEN1 was added together with RNase H1. ( C ) Schematic of template used in panel D. The downstream chimeric oligonucleotide was labelled at the 3′-end. The possible products are illustrated (b–d). ( D ) Coupled nuclease gap-filling ligation assay was performed in the presence of RNase H1 alone or together with FEN1 (35 fmol) as indicated. Lanes 2–4 contained only RNase H1 with or without FEN1 to monitor nuclease activity. RNase H1 cleaved the downstream oligonucleotide leaving 1–3 unprocessed ribonucleotides. FEN1 cleaved the remaining ribonucleotides, resulting in a shorter product. POLγ and DNA ligase (ligase III or T4 DNA ligase) were added in lanes 5–10. Ligase III showed ligation only when both RNase H1 and FEN1 were added. However, T4 DNA ligase could ligate without FEN1. Lanes 12–14 are ligation controls using a 3′-end labelled DNA-only oligonucleotide with a 5′-end phosphate. Note that the short band (

    Article Snippet: The reactions were incubated at 37°C for 30 min, after which 1 U of T4 DNA ligase or 300 fmol DNA ligase III was added.

    Techniques: Ligation, Activity Assay

    RNase H1 processing coupled to POLγ dependent DNA synthesis does not produce ligatable nicks. ( A ) Schematic of the coupled nuclease gap-filling ligation assay performed on a gapped OriL substrate (a). The upstream oligonucleotide was radioactively labelled at the 5′-end. The possible products are illustrated (b–d). (–) Coupled nuclease gap-filling ligation assay as shown in A. POLγ filled the gap (lane 2, marked b) and had limited strand displacement activity (SD). Note though that POLγ completely displaces the downstream oligonucleotide in a small fraction of templates (80 nt band, lanes 3–6,), RNase H1 cleaved the RNA in the substrate (lane 3), enabling further gap-filling (marked c). Only very low levels of ligated products were formed in the presence of 80–320 fmol DNA ligase III (lanes 4–6). A prominent 80 nt ligated product was formed with T4 DNA ligase (lane 7, marked d). The letters a-d correspond to the illustrations in panel A. ( C ) Ligation assay on a nicked substrate containing RNA tracts of varying length downstream of the nick in the presence of 80–320 fmol DNA ligase III. DNA ligase III discriminates against nicked substrates that contain increasing stretches of ribonucleotides. Two, but not five or more ribonucleotides, can be ligated. ( D ) As in C, except performed with T4 ligase (1–8 U). T4 ligase can ligate five but not 10 ribonucleotides. ( E ) T4 ligase-mediated ligation is abolished in the presence of the mutant RNase H1 proteins. The letters a-d correspond to the illustrations in panel A.

    Journal: Nucleic Acids Research

    Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL

    doi: 10.1093/nar/gky708

    Figure Lengend Snippet: RNase H1 processing coupled to POLγ dependent DNA synthesis does not produce ligatable nicks. ( A ) Schematic of the coupled nuclease gap-filling ligation assay performed on a gapped OriL substrate (a). The upstream oligonucleotide was radioactively labelled at the 5′-end. The possible products are illustrated (b–d). (–) Coupled nuclease gap-filling ligation assay as shown in A. POLγ filled the gap (lane 2, marked b) and had limited strand displacement activity (SD). Note though that POLγ completely displaces the downstream oligonucleotide in a small fraction of templates (80 nt band, lanes 3–6,), RNase H1 cleaved the RNA in the substrate (lane 3), enabling further gap-filling (marked c). Only very low levels of ligated products were formed in the presence of 80–320 fmol DNA ligase III (lanes 4–6). A prominent 80 nt ligated product was formed with T4 DNA ligase (lane 7, marked d). The letters a-d correspond to the illustrations in panel A. ( C ) Ligation assay on a nicked substrate containing RNA tracts of varying length downstream of the nick in the presence of 80–320 fmol DNA ligase III. DNA ligase III discriminates against nicked substrates that contain increasing stretches of ribonucleotides. Two, but not five or more ribonucleotides, can be ligated. ( D ) As in C, except performed with T4 ligase (1–8 U). T4 ligase can ligate five but not 10 ribonucleotides. ( E ) T4 ligase-mediated ligation is abolished in the presence of the mutant RNase H1 proteins. The letters a-d correspond to the illustrations in panel A.

    Article Snippet: The reactions were incubated at 37°C for 30 min, after which 1 U of T4 DNA ligase or 300 fmol DNA ligase III was added.

    Techniques: DNA Synthesis, Ligation, Activity Assay, Mutagenesis

    YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with T4 DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .

    Journal: Cell

    Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops

    doi: 10.1016/j.cell.2017.11.008

    Figure Lengend Snippet: YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with T4 DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .

    Article Snippet: YY1: 0.25 nM DNA, 1× T4 DNA ligase buffer (NEB B0202S), H2 O 0.12 μg/μL of YY1.

    Techniques: In Vitro, Nucleic Acid Electrophoresis, DNA Ligation, Incubation

    Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.

    Journal: Langmuir : the ACS journal of surfaces and colloids

    Article Title: Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains

    doi: 10.1021/acs.langmuir.8b01812

    Figure Lengend Snippet: Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.

    Article Snippet: PAGE-purified oligos were purchased from IDT. ssDNA circles were prepared by annealing 5 μ M phosphorylated template oligo (/5Phos/AG GAG AAA AAG AAA AAA AGA AAA GAA GG) and 4.5 μ M biotinylated primer oligo (5/Biosg/TC TCC TCC TTC T) in 1× T4 ligase reaction buffer (NEB B0202S)., Oligos were heated to 75 °C for 5 min and cooled to 4 °C at a rate of −1 °C min−1 .

    Techniques: Synthesized, End Labeling, Labeling, Recombinase Polymerase Amplification, Flow Cytometry