t4 dna ligase reaction buffer  (New England Biolabs)


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    Structured Review

    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 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "YY1 Is a Structural Regulator of Enhancer-Promoter Loops"

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

    Journal: Cell

    doi: 10.1016/j.cell.2017.11.008

    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. .
    Figure Legend 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. .

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

    2) Product Images from "Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains"

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

    Journal: Langmuir : the ACS journal of surfaces and colloids

    doi: 10.1021/acs.langmuir.8b01812

    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.
    Figure Legend 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.

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

    3) Product Images from "Functional studies of genetic variants associated with human diseases in Notch signaling-related genes using Drosophila"

    Article Title: Functional studies of genetic variants associated with human diseases in Notch signaling-related genes using Drosophila

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-0716-2201-8_19

    Generation of an amx-yellow[wing2+] homology directed repair donor construct using the Golden Gate cloning strategy. (A) In order to clone the upstream (UHA) and downstream homology arms (DHA) of the homology directed repair (HDR) donor construct, perform PCR using specific primers and genomic fly DNA. In addition to the segments that anneal with the genomic DNA, the primers designed here have features that facilitate the subcloning of these fragments using the Golden Gate strategy. (B) In addition to the two homology arms generated by PCR, this protocol requires two plasmids, one that provides the vector backbone of the final product (pBH vector shown on the left) and another that provides the yellow[wing2+] cassette. (C) Assembly of the amx-yellow[wing2+] HDR plasmid through the Golden Gate reaction. By mixing the UHA and DHA from (A) , the two plasmids from (B) , a type IIs restriction enzyme BsaI and a DNA ligase, the four segments will be assembled into one plasmid through repetitive digestion and ligation reactions based on the specific overhangs created by the BsaI digestion (shown as overhangs ① to ④).
    Figure Legend Snippet: Generation of an amx-yellow[wing2+] homology directed repair donor construct using the Golden Gate cloning strategy. (A) In order to clone the upstream (UHA) and downstream homology arms (DHA) of the homology directed repair (HDR) donor construct, perform PCR using specific primers and genomic fly DNA. In addition to the segments that anneal with the genomic DNA, the primers designed here have features that facilitate the subcloning of these fragments using the Golden Gate strategy. (B) In addition to the two homology arms generated by PCR, this protocol requires two plasmids, one that provides the vector backbone of the final product (pBH vector shown on the left) and another that provides the yellow[wing2+] cassette. (C) Assembly of the amx-yellow[wing2+] HDR plasmid through the Golden Gate reaction. By mixing the UHA and DHA from (A) , the two plasmids from (B) , a type IIs restriction enzyme BsaI and a DNA ligase, the four segments will be assembled into one plasmid through repetitive digestion and ligation reactions based on the specific overhangs created by the BsaI digestion (shown as overhangs ① to ④).

    Techniques Used: Construct, Clone Assay, Polymerase Chain Reaction, Subcloning, Generated, Plasmid Preparation, Ligation

    4) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.
    Figure Legend Snippet: Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Techniques Used: Binding Assay

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    5) Product Images from "Assessing protein dynamics on low complexity single-strand DNA curtains"

    Article Title: Assessing protein dynamics on low complexity single-strand DNA curtains

    Journal: bioRxiv

    doi: 10.1101/376798

    Assembly of low complexity ssDNA curtains. (A) A phosphorylated template (black) and biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low complexity ssDNA comprised 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.
    Figure Legend Snippet: Assembly of low complexity ssDNA curtains. (A) A phosphorylated template (black) and biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low complexity ssDNA comprised 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.

    Techniques Used: Synthesized, End Labeling, Labeling, Recombinase Polymerase Amplification

    6) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.
    Figure Legend Snippet: Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Techniques Used: Binding Assay

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    7) Product Images from "Expanding luciferase reporter systems for cell-free protein expression"

    Article Title: Expanding luciferase reporter systems for cell-free protein expression

    Journal: bioRxiv

    doi: 10.1101/2022.05.10.491427

    The result of caffeic acid conversion assay 2. NPGA, Hisps, H3H, and Luz were individually expressed in TXTL. NPGA and Hisps TXTLs were first mixed with cofactors and substrates (caffeic acid or hispidin). The reaction was incubated at 37°C for 30 minutes to facilitate phosphopanthetheinylation. The aliquot of the reaction was taken for HPLC analysis after this incubation. Then, H3H and Luz TXTLs were added to the reaction and measured the luminescence at 25°C for 1 hour, every 5 minutes. The reaction with hispidin slightly generated light. The reaction with caffeic acid was as same as Control reaction. This indicates that the caffeic acid was not converted into hispidin. Control stands for a reaction contains TXTLs without enzyme expression.
    Figure Legend Snippet: The result of caffeic acid conversion assay 2. NPGA, Hisps, H3H, and Luz were individually expressed in TXTL. NPGA and Hisps TXTLs were first mixed with cofactors and substrates (caffeic acid or hispidin). The reaction was incubated at 37°C for 30 minutes to facilitate phosphopanthetheinylation. The aliquot of the reaction was taken for HPLC analysis after this incubation. Then, H3H and Luz TXTLs were added to the reaction and measured the luminescence at 25°C for 1 hour, every 5 minutes. The reaction with hispidin slightly generated light. The reaction with caffeic acid was as same as Control reaction. This indicates that the caffeic acid was not converted into hispidin. Control stands for a reaction contains TXTLs without enzyme expression.

    Techniques Used: Incubation, High Performance Liquid Chromatography, Generated, Expressing

    Characterization of substrate specificities (A) Schematic image of firefly luciferase (FLuc), renilla luciferase (RLuc), and NanoLuc luciferase (NanoLuc) reactions. FLuc oxidizes D-luciferin with ATP and Mg + to produce light. RLuc and NanoLuc oxidize coelenterazine h and furimazine, respectively, with ATP to produce light. (B) Luminescence measurement for substrate specificity assay for 5 luciferases. The luciferases (FLuc, RLuc, NanoLuc, H3H-Luz, and LuxAB-Fre) were expressed in TXTL. Then, the individual substrates (D-luciferin, coelenterazine h, furimazine, hispidin, and octanaldehyde) with corresponding co-factors were added to the reaction and measured its light emission without emission filters. Substrate concentrations were 10 μM, except 1 mM for octanaldehyde. (C-F) The substrate multiplexing assay. The substrate mixtures were prepared as “All” (D-luciferin, Coelenterazine h, hispidin, octanaldehyde, Mg + , ATP, NADPH, FMN) or “All minus one” that contains all except one that a substrate is supposed to react with a tested luciferase. The assay was performed by mixing substrates with TXTL expressing (C) FLuc, (D) RLuc, (E) H3H-Luz, or (F) LuxAB-Fre, and the luminescence was measured without emission filters. ATP, adenosine triphosphate. The graphs show means with error bars that signify SEM (n = 3).
    Figure Legend Snippet: Characterization of substrate specificities (A) Schematic image of firefly luciferase (FLuc), renilla luciferase (RLuc), and NanoLuc luciferase (NanoLuc) reactions. FLuc oxidizes D-luciferin with ATP and Mg + to produce light. RLuc and NanoLuc oxidize coelenterazine h and furimazine, respectively, with ATP to produce light. (B) Luminescence measurement for substrate specificity assay for 5 luciferases. The luciferases (FLuc, RLuc, NanoLuc, H3H-Luz, and LuxAB-Fre) were expressed in TXTL. Then, the individual substrates (D-luciferin, coelenterazine h, furimazine, hispidin, and octanaldehyde) with corresponding co-factors were added to the reaction and measured its light emission without emission filters. Substrate concentrations were 10 μM, except 1 mM for octanaldehyde. (C-F) The substrate multiplexing assay. The substrate mixtures were prepared as “All” (D-luciferin, Coelenterazine h, hispidin, octanaldehyde, Mg + , ATP, NADPH, FMN) or “All minus one” that contains all except one that a substrate is supposed to react with a tested luciferase. The assay was performed by mixing substrates with TXTL expressing (C) FLuc, (D) RLuc, (E) H3H-Luz, or (F) LuxAB-Fre, and the luminescence was measured without emission filters. ATP, adenosine triphosphate. The graphs show means with error bars that signify SEM (n = 3).

    Techniques Used: Luciferase, Multiplexing, Expressing

    Background signals of the substrate specificity assay. The luciferases used in the reaction are indicated as FLuc, RLuc, H3H-Luz, or LuxAB-Fre. Control stands for reaction without enzyme expression. Substrates in the reaction were indicated as “All” (D-luciferin, coelenterazine h, hispidin, octanaldehyde) or “All minus one”, that one is the substrate supposed to react with the tested luciferase.
    Figure Legend Snippet: Background signals of the substrate specificity assay. The luciferases used in the reaction are indicated as FLuc, RLuc, H3H-Luz, or LuxAB-Fre. Control stands for reaction without enzyme expression. Substrates in the reaction were indicated as “All” (D-luciferin, coelenterazine h, hispidin, octanaldehyde) or “All minus one”, that one is the substrate supposed to react with the tested luciferase.

    Techniques Used: Expressing, Luciferase

    H3H-Luz tested with burden-reduced TXTL and fusion proteins. (A) The schematic of how H3H-carrying TXTL works. The plasmid coding H3H gene under the sigma 70 promoter is transformed into E. coli Rosseta 2 strain. The cell-free extract is made with that strain; thus, the extract contains H3H. Once Luz is expressed in the TXTL, Luz produces luminescence by coordinating with H3H. (B) Luminescence measurement in the H3H pre-containing TXTL. Luz plasmids were incubated with hispidin at 30°C. The Luz plasmid containing reaction (yellow dots) generated light during the TXTL reaction, while the reaction without Luz plasmid did not (black squares.) (C) Luminescence measurement of the H3H-Luz system with eGFP fused Luz constructs. H3H and Luz proteins were expressed in TXTL. After the expression, hispidin and NADPH were added, followed by luminescence measurement. (D) Fluorescence measurement of the eGFP fused Luz. Luz proteins were expressed in TXTL and the fluorescence was measured. Luz, Luz luciferase without a fusion protein; N-GFP, N-terminal eGFP fusion with Luz; C-GFP, C-terminal eGFP fusion with Luz; Control, reaction without enzyme expression. The graphs show means with error bars that signify SEM (n = 3).
    Figure Legend Snippet: H3H-Luz tested with burden-reduced TXTL and fusion proteins. (A) The schematic of how H3H-carrying TXTL works. The plasmid coding H3H gene under the sigma 70 promoter is transformed into E. coli Rosseta 2 strain. The cell-free extract is made with that strain; thus, the extract contains H3H. Once Luz is expressed in the TXTL, Luz produces luminescence by coordinating with H3H. (B) Luminescence measurement in the H3H pre-containing TXTL. Luz plasmids were incubated with hispidin at 30°C. The Luz plasmid containing reaction (yellow dots) generated light during the TXTL reaction, while the reaction without Luz plasmid did not (black squares.) (C) Luminescence measurement of the H3H-Luz system with eGFP fused Luz constructs. H3H and Luz proteins were expressed in TXTL. After the expression, hispidin and NADPH were added, followed by luminescence measurement. (D) Fluorescence measurement of the eGFP fused Luz. Luz proteins were expressed in TXTL and the fluorescence was measured. Luz, Luz luciferase without a fusion protein; N-GFP, N-terminal eGFP fusion with Luz; C-GFP, C-terminal eGFP fusion with Luz; Control, reaction without enzyme expression. The graphs show means with error bars that signify SEM (n = 3).

    Techniques Used: Plasmid Preparation, Transformation Assay, Incubation, Generated, Construct, Expressing, Fluorescence, Luciferase

    Luminescence kinetics measurement with octanaldehyde. After expressing LuxABCDE+Fre or LuxAB+Fre in TXTL, 1 mM octanaldehyde was added as the substrate (time = 0). The luminescence was measured after 0.5, 1, 6, 8 hours. LuxABCDE-Fre, a reaction with TXTL expressing LuxAB-Fre and LuxCDE; LuxAB-Fre, a reaction with TXTL expressing LuxAB-Fre; Control, reaction with TXTL without enzyme expression.
    Figure Legend Snippet: Luminescence kinetics measurement with octanaldehyde. After expressing LuxABCDE+Fre or LuxAB+Fre in TXTL, 1 mM octanaldehyde was added as the substrate (time = 0). The luminescence was measured after 0.5, 1, 6, 8 hours. LuxABCDE-Fre, a reaction with TXTL expressing LuxAB-Fre and LuxCDE; LuxAB-Fre, a reaction with TXTL expressing LuxAB-Fre; Control, reaction with TXTL without enzyme expression.

    Techniques Used: Expressing

    Fluorescence generated from eGFP-luciferase fusion proteins with extended GS-linker. All fusion proteins were expressed in TXTL at 30 °C for 8 hours, followed by fluorescence measurement. 19 μl of TXTL was used for the measurement. eGFP and luciferases are linked through 3x GS-linker (GGGGS.) Control stands for a reaction without protein expression.
    Figure Legend Snippet: Fluorescence generated from eGFP-luciferase fusion proteins with extended GS-linker. All fusion proteins were expressed in TXTL at 30 °C for 8 hours, followed by fluorescence measurement. 19 μl of TXTL was used for the measurement. eGFP and luciferases are linked through 3x GS-linker (GGGGS.) Control stands for a reaction without protein expression.

    Techniques Used: Fluorescence, Generated, Luciferase, Expressing

    Substrate regeneration system with LuxABCDE-Fre (A) The schematic of the LuxABCDE-Fre substrate regeneration system. LuxAB generates light with reduced flavin mononucleotide (FMNH 2 ) and long-chain aldehyde; those substrates are converted into oxidized flavin mononucleotide (FMN) and corresponding long-chain acid. NAD(P)H-flavin reductase (Fre) reduces FMN back to FMNH 2 , and LuxCDE reduces the acid back to the corresponding aldehyde. (B) Luminescence measurement with the LuxABCDE-Fre system. LuxAB-Fre and LuxCDE were expressed in TXTL and mixed with 1 mM long-chain fatty acids (octanoic acid, decanoic acid, dodecanoic acid, or tetradecanoic acid) or caprylic aldehyde, followed by luminescence measurement. The reaction also contained FMN, NADPH, and ATP. Control represents a reaction using TXTL without enzyme expression. (C) Luminescence kinetics measurement with decanoic acid. LuxAB-Fre and LuxCDE were expressed in TXTL. For LuxABCDE-Fre reaction, the TXTL expressing LuxAB-Fre and LuxCDE were mixed with 1 mM decanoic acid (time = 0). For LuxAB-Fre reaction, the TXTL expressing LuxAB-Fre was used. For Control reaction, TXTL without enzyme expression was used. The reaction also contained FMN, NADPH, and ATP. The luminescence was measured after 0.5, 1, 6, and 8 hours. The graphs show means with error bars that signify SEM (n = 3).
    Figure Legend Snippet: Substrate regeneration system with LuxABCDE-Fre (A) The schematic of the LuxABCDE-Fre substrate regeneration system. LuxAB generates light with reduced flavin mononucleotide (FMNH 2 ) and long-chain aldehyde; those substrates are converted into oxidized flavin mononucleotide (FMN) and corresponding long-chain acid. NAD(P)H-flavin reductase (Fre) reduces FMN back to FMNH 2 , and LuxCDE reduces the acid back to the corresponding aldehyde. (B) Luminescence measurement with the LuxABCDE-Fre system. LuxAB-Fre and LuxCDE were expressed in TXTL and mixed with 1 mM long-chain fatty acids (octanoic acid, decanoic acid, dodecanoic acid, or tetradecanoic acid) or caprylic aldehyde, followed by luminescence measurement. The reaction also contained FMN, NADPH, and ATP. Control represents a reaction using TXTL without enzyme expression. (C) Luminescence kinetics measurement with decanoic acid. LuxAB-Fre and LuxCDE were expressed in TXTL. For LuxABCDE-Fre reaction, the TXTL expressing LuxAB-Fre and LuxCDE were mixed with 1 mM decanoic acid (time = 0). For LuxAB-Fre reaction, the TXTL expressing LuxAB-Fre was used. For Control reaction, TXTL without enzyme expression was used. The reaction also contained FMN, NADPH, and ATP. The luminescence was measured after 0.5, 1, 6, and 8 hours. The graphs show means with error bars that signify SEM (n = 3).

    Techniques Used: Expressing

    Characterization of H3H-Lux and LuxAB-Fre luciferase systems in TXTL. (A) Schematic of H3H-Luz luciferase reaction. Hispidin is converted to 3-hydroxyhispidin by hispidin-3-hydroxylase (H3H) and 3-hydroxyhispidin is oxidized and converted into a high energy intermediate by the luciferase (Luz). This intermediate decays into caffeylpyruvic acid with light emission. (B) The H3H-Luz luminescence measurement. H3H and Luz were expressed in TXTL. The luminescence was measured right after adding NADPH and hisipidin into the TXTL. (C) Schematic of LuxAB-Fre luciferase reaction. Oxidized flavin mononucleotide (FMN) is reduced into reduced flavin mononucleotide (FMNH 2 ) by NAD(P)H-flavin reductase (Fre). The luciferase (LuxAB) converts FMNH 2 and long-chain aldehydes into FMN and the corresponding long-chain acids with light emission. (E) ATP supplementation increased the light emission of LuxAB-Fre. Octanaldehyde was added as the substrate. (D) The LuxAB-Fre luminescence measurement with different long-chain fatty aldehydes. LuxA, LuxB and Fre were expressed in TXTL. The luminescence was measured right after adding FMN, NADPH, ATP, and substrates (octanaldehyde, decyl aldehyde, and dodecyl aldehyde.) NADPH, nicotinamide adenine dinucleotide phosphate; ATP, adenosine triphosphate; Control, reaction without enzyme expression. The graphs show means with error bars that signify SEM (n = 3).
    Figure Legend Snippet: Characterization of H3H-Lux and LuxAB-Fre luciferase systems in TXTL. (A) Schematic of H3H-Luz luciferase reaction. Hispidin is converted to 3-hydroxyhispidin by hispidin-3-hydroxylase (H3H) and 3-hydroxyhispidin is oxidized and converted into a high energy intermediate by the luciferase (Luz). This intermediate decays into caffeylpyruvic acid with light emission. (B) The H3H-Luz luminescence measurement. H3H and Luz were expressed in TXTL. The luminescence was measured right after adding NADPH and hisipidin into the TXTL. (C) Schematic of LuxAB-Fre luciferase reaction. Oxidized flavin mononucleotide (FMN) is reduced into reduced flavin mononucleotide (FMNH 2 ) by NAD(P)H-flavin reductase (Fre). The luciferase (LuxAB) converts FMNH 2 and long-chain aldehydes into FMN and the corresponding long-chain acids with light emission. (E) ATP supplementation increased the light emission of LuxAB-Fre. Octanaldehyde was added as the substrate. (D) The LuxAB-Fre luminescence measurement with different long-chain fatty aldehydes. LuxA, LuxB and Fre were expressed in TXTL. The luminescence was measured right after adding FMN, NADPH, ATP, and substrates (octanaldehyde, decyl aldehyde, and dodecyl aldehyde.) NADPH, nicotinamide adenine dinucleotide phosphate; ATP, adenosine triphosphate; Control, reaction without enzyme expression. The graphs show means with error bars that signify SEM (n = 3).

    Techniques Used: Luciferase, Expressing

    LuxA and LuxB capability as fusion proteins. (A and B) eGFP fluorescence measurement in TXTL. Fusion LuxA and LuxB were expressed in TXTL at 30°C for 8 hours, followed by the fluorescence measurement. (C) Luminescence measurement with all the combinations of LuxA and LuxB fusion constructs. LuxA and LuxB were expressed in TXTL, and then 1 mM Octanaldehyde was added, followed by luminescence measurement. Green shading behind the construct images indicates that the constructs fluorescence when expressed in TXTL. N-GFP, N-terminal eGFP fusion luciferase; C-GFP, C-terminal eGFP fusion luciferase; Control, reaction without enzyme expression.
    Figure Legend Snippet: LuxA and LuxB capability as fusion proteins. (A and B) eGFP fluorescence measurement in TXTL. Fusion LuxA and LuxB were expressed in TXTL at 30°C for 8 hours, followed by the fluorescence measurement. (C) Luminescence measurement with all the combinations of LuxA and LuxB fusion constructs. LuxA and LuxB were expressed in TXTL, and then 1 mM Octanaldehyde was added, followed by luminescence measurement. Green shading behind the construct images indicates that the constructs fluorescence when expressed in TXTL. N-GFP, N-terminal eGFP fusion luciferase; C-GFP, C-terminal eGFP fusion luciferase; Control, reaction without enzyme expression.

    Techniques Used: Fluorescence, Construct, Luciferase, Expressing

    HiBiT reporter system (A) The schematic of how LgBiT-carrying E. coli cell-free extract works. The plasmid coding LgBiT gene under the sigma 70 promoter is transformed into E. coli Rosetta 2 strain. The cell-free extract is made with that strain; thus, the extract contains LgBiT. Once HiBiT is expressed in TXTL, HiBiT produces luminescence by reconstituting full luciferase with LgBiT. (B) eGFP fluorescence measurement of HiBiT-GFP fusion proteins. Fusion proteins were expressed in TXTL at 30°C for 8 hours, followed by the measurement. (C) End-point luminescence assay with fusion HiBiTs. The fusion HiBiTs were expressed in LgBiT-containing TXTL at 30°C for 8 hours. After the expression, 1 µM Furimazine was added, followed by luminescence measurement. (D) Luminescence kinetics measurement with LgBiT-containing TXTL. HiBiT plasmids and 1 µM Furimazine were added at the TXTL reaction set up and incubated at 30°C. The luminescence was measured every 5 minutes during the TXTL reaction. N-GFP, N-terminal eGFP fusion with HiBiT; C-GFP, C-terminal eGFP fusion with HiBiT; Control, reaction without enzyme expression. The graphs show means with error bars that signify SEM (n = 3).
    Figure Legend Snippet: HiBiT reporter system (A) The schematic of how LgBiT-carrying E. coli cell-free extract works. The plasmid coding LgBiT gene under the sigma 70 promoter is transformed into E. coli Rosetta 2 strain. The cell-free extract is made with that strain; thus, the extract contains LgBiT. Once HiBiT is expressed in TXTL, HiBiT produces luminescence by reconstituting full luciferase with LgBiT. (B) eGFP fluorescence measurement of HiBiT-GFP fusion proteins. Fusion proteins were expressed in TXTL at 30°C for 8 hours, followed by the measurement. (C) End-point luminescence assay with fusion HiBiTs. The fusion HiBiTs were expressed in LgBiT-containing TXTL at 30°C for 8 hours. After the expression, 1 µM Furimazine was added, followed by luminescence measurement. (D) Luminescence kinetics measurement with LgBiT-containing TXTL. HiBiT plasmids and 1 µM Furimazine were added at the TXTL reaction set up and incubated at 30°C. The luminescence was measured every 5 minutes during the TXTL reaction. N-GFP, N-terminal eGFP fusion with HiBiT; C-GFP, C-terminal eGFP fusion with HiBiT; Control, reaction without enzyme expression. The graphs show means with error bars that signify SEM (n = 3).

    Techniques Used: Plasmid Preparation, Transformation Assay, Luciferase, Fluorescence, Luminescence Assay, Expressing, Incubation

    Western blot gels from the substrate specificity reactions. The loaded samples directly came from the reactions shown in Fig. 2B , indicating that the luciferase enzymes were expressed in the reactions. 15 μl of TxTL was loaded on each lane. FLuc and RLuc samples were fractionated on a 7.5% gel for 70 minutes at 100V. NanoLuc and H3H-Luz samples were fractionated on a 12% gel for 80 minutes at 100V. M, BLUEstain 2 Protein ladder, 5-245 kDa (Goldbio, P008-500); substrate names, a substrate that is contained in the reaction; T7RNAP, N-terminal His-tagged T7 RNA polymerase; FLuc, firefly luciferase with C-terminal His-tag; RLuc, Renilla luciferase with C-terminal His-tag; NanoLuc, NanoLuc luciferase with C-terminal His-Tag; H3H, hispidin-3-hydroxylase with C-terminal His-tag; Luz, fungi luciferase with C-terminal His-tag.
    Figure Legend Snippet: Western blot gels from the substrate specificity reactions. The loaded samples directly came from the reactions shown in Fig. 2B , indicating that the luciferase enzymes were expressed in the reactions. 15 μl of TxTL was loaded on each lane. FLuc and RLuc samples were fractionated on a 7.5% gel for 70 minutes at 100V. NanoLuc and H3H-Luz samples were fractionated on a 12% gel for 80 minutes at 100V. M, BLUEstain 2 Protein ladder, 5-245 kDa (Goldbio, P008-500); substrate names, a substrate that is contained in the reaction; T7RNAP, N-terminal His-tagged T7 RNA polymerase; FLuc, firefly luciferase with C-terminal His-tag; RLuc, Renilla luciferase with C-terminal His-tag; NanoLuc, NanoLuc luciferase with C-terminal His-Tag; H3H, hispidin-3-hydroxylase with C-terminal His-tag; Luz, fungi luciferase with C-terminal His-tag.

    Techniques Used: Western Blot, Luciferase

    The result of caffeic acid con version assay 1 NPGA, Hisps, H3H, and Luz were individually expressed in TXTL and mixed with cofactors and substrates (caffeic acid or hispidin). The luminescence was measured at 25°C for 1 hour, every 5 minutes. While the reaction with hispidin generated light, the reaction with caffeic acid did not. This indicates that the caffeic acid was not converted into hispidin. Control contains TXTLs without enzyme expression.
    Figure Legend Snippet: The result of caffeic acid con version assay 1 NPGA, Hisps, H3H, and Luz were individually expressed in TXTL and mixed with cofactors and substrates (caffeic acid or hispidin). The luminescence was measured at 25°C for 1 hour, every 5 minutes. While the reaction with hispidin generated light, the reaction with caffeic acid did not. This indicates that the caffeic acid was not converted into hispidin. Control contains TXTLs without enzyme expression.

    Techniques Used: Generated, Expressing

    8) Product Images from "YY1 Is a Structural Regulator of Enhancer-Promoter Loops"

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

    Journal: Cell

    doi: 10.1016/j.cell.2017.11.008

    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. .
    Figure Legend 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. .

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

    9) Product Images from "Functional studies of genetic variants associated with human diseases in Notch signaling-related genes using Drosophila"

    Article Title: Functional studies of genetic variants associated with human diseases in Notch signaling-related genes using Drosophila

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-0716-2201-8_19

    Generation of an amx-yellow[wing2+] homology directed repair donor construct using the Golden Gate cloning strategy. (A) In order to clone the upstream (UHA) and downstream homology arms (DHA) of the homology directed repair (HDR) donor construct, perform PCR using specific primers and genomic fly DNA. In addition to the segments that anneal with the genomic DNA, the primers designed here have features that facilitate the subcloning of these fragments using the Golden Gate strategy. (B) In addition to the two homology arms generated by PCR, this protocol requires two plasmids, one that provides the vector backbone of the final product (pBH vector shown on the left) and another that provides the yellow[wing2+] cassette. (C) Assembly of the amx-yellow[wing2+] HDR plasmid through the Golden Gate reaction. By mixing the UHA and DHA from (A) , the two plasmids from (B) , a type IIs restriction enzyme BsaI and a DNA ligase, the four segments will be assembled into one plasmid through repetitive digestion and ligation reactions based on the specific overhangs created by the BsaI digestion (shown as overhangs ① to ④).
    Figure Legend Snippet: Generation of an amx-yellow[wing2+] homology directed repair donor construct using the Golden Gate cloning strategy. (A) In order to clone the upstream (UHA) and downstream homology arms (DHA) of the homology directed repair (HDR) donor construct, perform PCR using specific primers and genomic fly DNA. In addition to the segments that anneal with the genomic DNA, the primers designed here have features that facilitate the subcloning of these fragments using the Golden Gate strategy. (B) In addition to the two homology arms generated by PCR, this protocol requires two plasmids, one that provides the vector backbone of the final product (pBH vector shown on the left) and another that provides the yellow[wing2+] cassette. (C) Assembly of the amx-yellow[wing2+] HDR plasmid through the Golden Gate reaction. By mixing the UHA and DHA from (A) , the two plasmids from (B) , a type IIs restriction enzyme BsaI and a DNA ligase, the four segments will be assembled into one plasmid through repetitive digestion and ligation reactions based on the specific overhangs created by the BsaI digestion (shown as overhangs ① to ④).

    Techniques Used: Construct, Clone Assay, Polymerase Chain Reaction, Subcloning, Generated, Plasmid Preparation, Ligation

    10) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.
    Figure Legend Snippet: Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Techniques Used: Binding Assay

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    11) Product Images from "Universal Loop assembly (uLoop): open, efficient, and species-agnostic DNA fabrication"

    Article Title: Universal Loop assembly (uLoop): open, efficient, and species-agnostic DNA fabrication

    Journal: bioRxiv

    doi: 10.1101/744854

    Large-scale DNA assembly. Four L3 parts were assembled in the absence of a receiver plasmid through a SapI-mediated Loop assembly reaction and products were analysed by pulsed-field gel electrophoresis. Lane headings: M, Midrange PFG marker. C, control reaction (L3 parts digested with SapI). Assembly reaction using 1x (10 U µL-1) or 2x (20 U µL-1) T4 DNA ligase. Image shown corresponds to an inverted photograph of the gel with adjusted contrast. The white arrow indicates the monomeric fragments, the blue arrow indicates the dimeric composites, the red arrow indicates the trimeric composites and the green arrow indicates the tetrameric full-length assembly.
    Figure Legend Snippet: Large-scale DNA assembly. Four L3 parts were assembled in the absence of a receiver plasmid through a SapI-mediated Loop assembly reaction and products were analysed by pulsed-field gel electrophoresis. Lane headings: M, Midrange PFG marker. C, control reaction (L3 parts digested with SapI). Assembly reaction using 1x (10 U µL-1) or 2x (20 U µL-1) T4 DNA ligase. Image shown corresponds to an inverted photograph of the gel with adjusted contrast. The white arrow indicates the monomeric fragments, the blue arrow indicates the dimeric composites, the red arrow indicates the trimeric composites and the green arrow indicates the tetrameric full-length assembly.

    Techniques Used: Plasmid Preparation, Pulsed-Field Gel, Electrophoresis, Marker

    12) Product Images from "Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples"

    Article Title: Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples

    Journal: bioRxiv

    doi: 10.1101/2020.01.22.915009

    Average percentage of sequences carrying tag-jumps across amplicon pools built into Illumina libraries with four different library protocol treatments; +/+: T4 DNA polymerase blunt-ending and post-ligation PCR; −/+: no T4 DNA polymerase blunt-ending, with post-ligation PCR; +/−: T4 DNA polymerase blunt-ending and no post-ligation PCR; −/−: no T4 DNA polymerase blunt-ending and no post-ligation PCR (Tagsteady protocol) (n=6 for +/+, −/+, +/−, −/−). To mimic the effect of large amounts of single-stranded DNA generated in the metabarcoding PCR, aliquots of four of the amplicon pools were denatured and subsequently re-hybridized to form double-stranded DNA. These were then built into libraries with the −/− and +/− protocols (n=4 for d+/− and d−/−. Asterisks (*) denotes statistical significant difference between treatments (unpaired t-test, α=0.05).
    Figure Legend Snippet: Average percentage of sequences carrying tag-jumps across amplicon pools built into Illumina libraries with four different library protocol treatments; +/+: T4 DNA polymerase blunt-ending and post-ligation PCR; −/+: no T4 DNA polymerase blunt-ending, with post-ligation PCR; +/−: T4 DNA polymerase blunt-ending and no post-ligation PCR; −/−: no T4 DNA polymerase blunt-ending and no post-ligation PCR (Tagsteady protocol) (n=6 for +/+, −/+, +/−, −/−). To mimic the effect of large amounts of single-stranded DNA generated in the metabarcoding PCR, aliquots of four of the amplicon pools were denatured and subsequently re-hybridized to form double-stranded DNA. These were then built into libraries with the −/− and +/− protocols (n=4 for d+/− and d−/−. Asterisks (*) denotes statistical significant difference between treatments (unpaired t-test, α=0.05).

    Techniques Used: Amplification, Ligation, Polymerase Chain Reaction, Generated

    Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).
    Figure Legend Snippet: Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).

    Techniques Used: Polymerase Chain Reaction, Activity Assay, Sequencing, Ligation, Amplification

    Experimental overview. 1A) Six pools of 5’ twin-tagged amplicons generated with three metabarcoding primer sets were used to assess the effect of blunt-ending and post-ligation PCR on tag-jumps. Each of the six amplicon pools were subjected to four different treatments. 1B) The four treatments represent combinations with and without T4 DNA Polymerase blunt-ending in the end-repair step and 1C) with and without post-ligation PCR. 1D) This resulted in 24 libraries for the 6 amplicon pools, representing four library preparation treatments for each amplicon pool. 2A) To further assess the effect of T4 DNA Polymerase blunt-ending on the prevalence of tag-jumps, we denatured and re-hybridised four amplicon pools (16sMam1/2 and 16sIns1/2). 2B) End-repair was carried out with and without T4 DNA Polymerase blunt-ending and with no post-ligation PCR (2C). 3) Finally, to validate the robustness and stability of the Tagsteady protocol, we applied it to 15 pools of twin-tagged amplicons.
    Figure Legend Snippet: Experimental overview. 1A) Six pools of 5’ twin-tagged amplicons generated with three metabarcoding primer sets were used to assess the effect of blunt-ending and post-ligation PCR on tag-jumps. Each of the six amplicon pools were subjected to four different treatments. 1B) The four treatments represent combinations with and without T4 DNA Polymerase blunt-ending in the end-repair step and 1C) with and without post-ligation PCR. 1D) This resulted in 24 libraries for the 6 amplicon pools, representing four library preparation treatments for each amplicon pool. 2A) To further assess the effect of T4 DNA Polymerase blunt-ending on the prevalence of tag-jumps, we denatured and re-hybridised four amplicon pools (16sMam1/2 and 16sIns1/2). 2B) End-repair was carried out with and without T4 DNA Polymerase blunt-ending and with no post-ligation PCR (2C). 3) Finally, to validate the robustness and stability of the Tagsteady protocol, we applied it to 15 pools of twin-tagged amplicons.

    Techniques Used: Generated, Ligation, Polymerase Chain Reaction, Amplification

    13) Product Images from "YY1 Is a Structural Regulator of Enhancer-Promoter Loops"

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

    Journal: Cell

    doi: 10.1016/j.cell.2017.11.008

    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. .
    Figure Legend 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. .

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

    14) Product Images from "Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination"

    Article Title: Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2017.03.011

    Nucleoprotein filament dynamics on low sequence complexity ssDNA curtains. (A) Sequences of the two ssDNA oligonucleotides used for rolling circle replication. (B) Schematic of rolling circle replication (RCR) reaction. T4 DNA ligase ligates the template oligo to form a contiguous template strand. Next, phi29 DNA polymerase catalyzes the synthesis of long ssDNA molecules. (C) Agarose gel of several time points along the RCR synthesis reaction. The primer oligonucleotide was 32 P labeled on the 5 ′ -terminus phosphate ( gold star ). (D) Wide-field image of a microfabricated barrier set with double-tethered ssDNA curtains coated with RPA-TagRFP ( magenta ). Arrows and circles denote chromium barriers and pedestals, respectively. (E) Illustration and kymograph showing a single ssDNA molecule coated with ATTO488-RAD51(C319S) ( green ) replaced by RPA-TagRFP ( magenta ). Yellow dashed line denotes the injection of RPA–TagRFP into the flowcell. Buffer controls indicate when the buffer flow was toggled off and on to show that the florescent proteins retract to the Cr barriers simultaneously with the ssDNA molecule. This indicates that RAD51 and RPA are on the ssDNA molecule. Panel A: Adapted from Lee, K. S., Marciel, A. B., Kozlov, A. G., Schroeder, C. M., Lohman, T. M., Ha, T. (2014). Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. Journal of Molecular Biology, 426 , 2413 – 2421.
    Figure Legend Snippet: Nucleoprotein filament dynamics on low sequence complexity ssDNA curtains. (A) Sequences of the two ssDNA oligonucleotides used for rolling circle replication. (B) Schematic of rolling circle replication (RCR) reaction. T4 DNA ligase ligates the template oligo to form a contiguous template strand. Next, phi29 DNA polymerase catalyzes the synthesis of long ssDNA molecules. (C) Agarose gel of several time points along the RCR synthesis reaction. The primer oligonucleotide was 32 P labeled on the 5 ′ -terminus phosphate ( gold star ). (D) Wide-field image of a microfabricated barrier set with double-tethered ssDNA curtains coated with RPA-TagRFP ( magenta ). Arrows and circles denote chromium barriers and pedestals, respectively. (E) Illustration and kymograph showing a single ssDNA molecule coated with ATTO488-RAD51(C319S) ( green ) replaced by RPA-TagRFP ( magenta ). Yellow dashed line denotes the injection of RPA–TagRFP into the flowcell. Buffer controls indicate when the buffer flow was toggled off and on to show that the florescent proteins retract to the Cr barriers simultaneously with the ssDNA molecule. This indicates that RAD51 and RPA are on the ssDNA molecule. Panel A: Adapted from Lee, K. S., Marciel, A. B., Kozlov, A. G., Schroeder, C. M., Lohman, T. M., Ha, T. (2014). Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. Journal of Molecular Biology, 426 , 2413 – 2421.

    Techniques Used: Sequencing, Agarose Gel Electrophoresis, Labeling, Recombinase Polymerase Amplification, Injection, Flow Cytometry

    15) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.
    Figure Legend Snippet: Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Techniques Used: Binding Assay

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    16) Product Images from "Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins"

    Article Title: Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa270

    Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.
    Figure Legend Snippet: Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.

    Techniques Used: Modification, Derivative Assay, Fluorescence, Hybridization, Ligation, Polymerase Chain Reaction, Generated, Amplification, Sequencing, Construct

    17) Product Images from "Functional studies of genetic variants associated with human diseases in Notch signaling-related genes using Drosophila"

    Article Title: Functional studies of genetic variants associated with human diseases in Notch signaling-related genes using Drosophila

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-0716-2201-8_19

    Generation of an amx-yellow[wing2+] homology directed repair donor construct using the Golden Gate cloning strategy. (A) In order to clone the upstream (UHA) and downstream homology arms (DHA) of the homology directed repair (HDR) donor construct, perform PCR using specific primers and genomic fly DNA. In addition to the segments that anneal with the genomic DNA, the primers designed here have features that facilitate the subcloning of these fragments using the Golden Gate strategy. (B) In addition to the two homology arms generated by PCR, this protocol requires two plasmids, one that provides the vector backbone of the final product (pBH vector shown on the left) and another that provides the yellow[wing2+] cassette. (C) Assembly of the amx-yellow[wing2+] HDR plasmid through the Golden Gate reaction. By mixing the UHA and DHA from (A) , the two plasmids from (B) , a type IIs restriction enzyme BsaI and a DNA ligase, the four segments will be assembled into one plasmid through repetitive digestion and ligation reactions based on the specific overhangs created by the BsaI digestion (shown as overhangs ① to ④).
    Figure Legend Snippet: Generation of an amx-yellow[wing2+] homology directed repair donor construct using the Golden Gate cloning strategy. (A) In order to clone the upstream (UHA) and downstream homology arms (DHA) of the homology directed repair (HDR) donor construct, perform PCR using specific primers and genomic fly DNA. In addition to the segments that anneal with the genomic DNA, the primers designed here have features that facilitate the subcloning of these fragments using the Golden Gate strategy. (B) In addition to the two homology arms generated by PCR, this protocol requires two plasmids, one that provides the vector backbone of the final product (pBH vector shown on the left) and another that provides the yellow[wing2+] cassette. (C) Assembly of the amx-yellow[wing2+] HDR plasmid through the Golden Gate reaction. By mixing the UHA and DHA from (A) , the two plasmids from (B) , a type IIs restriction enzyme BsaI and a DNA ligase, the four segments will be assembled into one plasmid through repetitive digestion and ligation reactions based on the specific overhangs created by the BsaI digestion (shown as overhangs ① to ④).

    Techniques Used: Construct, Clone Assay, Polymerase Chain Reaction, Subcloning, Generated, Plasmid Preparation, Ligation

    18) Product Images from "Biochemical Properties of a Decoy Oligodeoxynucleotide Inhibitor of STAT3 Transcription Factor"

    Article Title: Biochemical Properties of a Decoy Oligodeoxynucleotide Inhibitor of STAT3 Transcription Factor

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19061608

    Ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D) is unaffected by biotinylation. ( A ) Structures of parental and biotinylated STAT3 decoy (S3D) and CS3D. ( B ) CS3D and biotinylated CS3D were incubated with T4 DNA ligase overnight, followed by electrophoresis on a urea/polyacrylamide gel and staining with SYBR Gold.
    Figure Legend Snippet: Ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D) is unaffected by biotinylation. ( A ) Structures of parental and biotinylated STAT3 decoy (S3D) and CS3D. ( B ) CS3D and biotinylated CS3D were incubated with T4 DNA ligase overnight, followed by electrophoresis on a urea/polyacrylamide gel and staining with SYBR Gold.

    Techniques Used: Ligation, Incubation, Electrophoresis, Staining

    Efficient ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D). ( A ) Schematic representation of CS3D ligation with T4 DNA ligase. The complementary segments of the single-stranded decoy molecule spontaneously self-anneal. Enzymatic ligation with T4 DNA ligase was used to complete cyclization. ( B ) Incubations were performed in the absence or presence of T4 DNA ligase overnight. Multiple identical ligations ( n = 5) were simultaneously performed. Samples from each reaction were then electrophoresed on a urea/polyacrylamide gel, stained with SYBR Gold, and quantified by densitometry.
    Figure Legend Snippet: Efficient ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D). ( A ) Schematic representation of CS3D ligation with T4 DNA ligase. The complementary segments of the single-stranded decoy molecule spontaneously self-anneal. Enzymatic ligation with T4 DNA ligase was used to complete cyclization. ( B ) Incubations were performed in the absence or presence of T4 DNA ligase overnight. Multiple identical ligations ( n = 5) were simultaneously performed. Samples from each reaction were then electrophoresed on a urea/polyacrylamide gel, stained with SYBR Gold, and quantified by densitometry.

    Techniques Used: Ligation, Staining

    19) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.
    Figure Legend Snippet: Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Techniques Used: Binding Assay

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    20) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.
    Figure Legend Snippet: Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Techniques Used: Binding Assay

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    21) Product Images from "Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins"

    Article Title: Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa270

    Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.
    Figure Legend Snippet: Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.

    Techniques Used: Modification, Derivative Assay, Fluorescence, Hybridization, Ligation, Polymerase Chain Reaction, Generated, Amplification, Sequencing, Construct

    22) Product Images from "RE-SELEX: restriction enzyme-based evolution of structure-switching aptamer biosensors †"

    Article Title: RE-SELEX: restriction enzyme-based evolution of structure-switching aptamer biosensors †

    Journal: Chemical Science

    doi: 10.1039/d1sc02715h

    Homogenous EcoRI-HF SELEX to generate structure-switching aptamers. (a) FAM-labeled DNA library having an N 40 random region was hybridized to capture strand and the complex digested with EcoRI-HF. The cleaved product (70 nt) was gel purified and full-length product (90 nt) was recovered through split ligation by T4 DNA ligase. The re-ligated library members were hybridized to free capture strand and incubated with the target. Active biosensor sequences were enriched by EcoRI-HF digestion followed by PCR amplification. ssDNA library was purified by 10% denaturing PAGE and carried on to subsequent selection rounds. (b) Chemical structure of kanamycin A. (c) Progression of EcoRI-HF SELEX to generate structure-switching aptamers to kanamycin A. Following digestion, cleavage products were monitored by 10% denaturing PAGE. Band intensity was used to quantify percent uncleaved.
    Figure Legend Snippet: Homogenous EcoRI-HF SELEX to generate structure-switching aptamers. (a) FAM-labeled DNA library having an N 40 random region was hybridized to capture strand and the complex digested with EcoRI-HF. The cleaved product (70 nt) was gel purified and full-length product (90 nt) was recovered through split ligation by T4 DNA ligase. The re-ligated library members were hybridized to free capture strand and incubated with the target. Active biosensor sequences were enriched by EcoRI-HF digestion followed by PCR amplification. ssDNA library was purified by 10% denaturing PAGE and carried on to subsequent selection rounds. (b) Chemical structure of kanamycin A. (c) Progression of EcoRI-HF SELEX to generate structure-switching aptamers to kanamycin A. Following digestion, cleavage products were monitored by 10% denaturing PAGE. Band intensity was used to quantify percent uncleaved.

    Techniques Used: Labeling, Purification, Ligation, Incubation, Polymerase Chain Reaction, Amplification, Polyacrylamide Gel Electrophoresis, Selection

    23) Product Images from "Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA"

    Article Title: Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA

    Journal: RNA Biology

    doi: 10.1080/15476286.2020.1777378

    A workflow of sample preparation for determination of positions of DNA cleavage sites produced by Cas nucleases in vitro. The cleaved DNA fragments generated by Cas nuclease during in vitro DNA cleavage reaction (Step I) are blunted using T4 PNK and T4 DNA polymerase (Step II). A-base is added to 3ʹ ends (Step III) for further ligation of Illumina NEBNext sequencing adaptors containing uridine (Step IV). Uridines are cleaved out using the NEB USER enzyme, which combines uracil DNA glycosylase and endonuclease VIII activity. Next, the samples are barcoded to produce DNA libraries ready for high throughput sequencing.
    Figure Legend Snippet: A workflow of sample preparation for determination of positions of DNA cleavage sites produced by Cas nucleases in vitro. The cleaved DNA fragments generated by Cas nuclease during in vitro DNA cleavage reaction (Step I) are blunted using T4 PNK and T4 DNA polymerase (Step II). A-base is added to 3ʹ ends (Step III) for further ligation of Illumina NEBNext sequencing adaptors containing uridine (Step IV). Uridines are cleaved out using the NEB USER enzyme, which combines uracil DNA glycosylase and endonuclease VIII activity. Next, the samples are barcoded to produce DNA libraries ready for high throughput sequencing.

    Techniques Used: Sample Prep, Produced, In Vitro, Generated, Ligation, Sequencing, Activity Assay, Next-Generation Sequencing

    24) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.
    Figure Legend Snippet: Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Techniques Used: Binding Assay

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    25) Product Images from "Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins"

    Article Title: Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa270

    Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.
    Figure Legend Snippet: Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.

    Techniques Used: Modification, Derivative Assay, Fluorescence, Hybridization, Ligation, Polymerase Chain Reaction, Generated, Amplification, Sequencing, Construct

    26) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.
    Figure Legend Snippet: Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Techniques Used: Binding Assay

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    27) Product Images from "Biochemical Properties of a Decoy Oligodeoxynucleotide Inhibitor of STAT3 Transcription Factor"

    Article Title: Biochemical Properties of a Decoy Oligodeoxynucleotide Inhibitor of STAT3 Transcription Factor

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19061608

    Ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D) is unaffected by biotinylation. ( A ) Structures of parental and biotinylated STAT3 decoy (S3D) and CS3D. ( B ) CS3D and biotinylated CS3D were incubated with T4 DNA ligase overnight, followed by electrophoresis on a urea/polyacrylamide gel and staining with SYBR Gold.
    Figure Legend Snippet: Ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D) is unaffected by biotinylation. ( A ) Structures of parental and biotinylated STAT3 decoy (S3D) and CS3D. ( B ) CS3D and biotinylated CS3D were incubated with T4 DNA ligase overnight, followed by electrophoresis on a urea/polyacrylamide gel and staining with SYBR Gold.

    Techniques Used: Ligation, Incubation, Electrophoresis, Staining

    Efficient ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D). ( A ) Schematic representation of CS3D ligation with T4 DNA ligase. The complementary segments of the single-stranded decoy molecule spontaneously self-anneal. Enzymatic ligation with T4 DNA ligase was used to complete cyclization. ( B ) Incubations were performed in the absence or presence of T4 DNA ligase overnight. Multiple identical ligations ( n = 5) were simultaneously performed. Samples from each reaction were then electrophoresed on a urea/polyacrylamide gel, stained with SYBR Gold, and quantified by densitometry.
    Figure Legend Snippet: Efficient ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D). ( A ) Schematic representation of CS3D ligation with T4 DNA ligase. The complementary segments of the single-stranded decoy molecule spontaneously self-anneal. Enzymatic ligation with T4 DNA ligase was used to complete cyclization. ( B ) Incubations were performed in the absence or presence of T4 DNA ligase overnight. Multiple identical ligations ( n = 5) were simultaneously performed. Samples from each reaction were then electrophoresed on a urea/polyacrylamide gel, stained with SYBR Gold, and quantified by densitometry.

    Techniques Used: Ligation, Staining

    28) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.
    Figure Legend Snippet: Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Techniques Used: Binding Assay

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    29) Product Images from "Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins"

    Article Title: Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa270

    Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.
    Figure Legend Snippet: Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.

    Techniques Used: Modification, Derivative Assay, Fluorescence, Hybridization, Ligation, Polymerase Chain Reaction, Generated, Amplification, Sequencing, Construct

    30) Product Images from "CRISPR Toolbox for Genome Editing in Dictyostelium"

    Article Title: CRISPR Toolbox for Genome Editing in Dictyostelium

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2021.721630

    Single cloning step of a dual sgRNA expression vector. Annealed oligonucleotides are ligated to the tRNA–sgRNA junctions via Golden Gate reaction. DNA sequences of BpiI sites are indicated in yellow box. As the BpiI site is non-palindromic, re-digestion/ligation is not possible. Red and blue letters show the overhangs for cloning and target sequences of sgRNAs, respectively. CRISPR vector, T4 DNA ligase and annealed oligonucleotides for the first and second targets are mixed in a single tube to integrate two targets.
    Figure Legend Snippet: Single cloning step of a dual sgRNA expression vector. Annealed oligonucleotides are ligated to the tRNA–sgRNA junctions via Golden Gate reaction. DNA sequences of BpiI sites are indicated in yellow box. As the BpiI site is non-palindromic, re-digestion/ligation is not possible. Red and blue letters show the overhangs for cloning and target sequences of sgRNAs, respectively. CRISPR vector, T4 DNA ligase and annealed oligonucleotides for the first and second targets are mixed in a single tube to integrate two targets.

    Techniques Used: Clone Assay, Expressing, Plasmid Preparation, Ligation, CRISPR

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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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    New England Biolabs dna ligase
    Southern blots of catalase-defective mutants. Shown are the Southern blots of Hin <t>dIII-digested</t> genomic <t>DNA</t> from the wild type, Y4Nal (lane 2), and catalase-negative mutants, Aa1393 (lane 3), Aa1394 (lane 4), and Aa1395 (lane 5). Lane 1 contains digoxigenin-labeled DNA molecular weight markers (shown in kilobases) (Boehringer Mannheim). Probes were digoxigenin-labeled DNA containing the kan gene (A) or the katA gene (B).
    Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    Generation of an amx-yellow[wing2+] homology directed repair donor construct using the Golden Gate cloning strategy. (A) In order to clone the upstream (UHA) and downstream homology arms (DHA) of the homology directed repair (HDR) donor construct, perform PCR using specific primers and genomic fly DNA. In addition to the segments that anneal with the genomic DNA, the primers designed here have features that facilitate the subcloning of these fragments using the Golden Gate strategy. (B) In addition to the two homology arms generated by PCR, this protocol requires two plasmids, one that provides the vector backbone of the final product (pBH vector shown on the left) and another that provides the yellow[wing2+] cassette. (C) Assembly of the amx-yellow[wing2+] HDR plasmid through the Golden Gate reaction. By mixing the UHA and DHA from (A) , the two plasmids from (B) , a type IIs restriction enzyme BsaI and a DNA ligase, the four segments will be assembled into one plasmid through repetitive digestion and ligation reactions based on the specific overhangs created by the BsaI digestion (shown as overhangs ① to ④).

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Functional studies of genetic variants associated with human diseases in Notch signaling-related genes using Drosophila

    doi: 10.1007/978-1-0716-2201-8_19

    Figure Lengend Snippet: Generation of an amx-yellow[wing2+] homology directed repair donor construct using the Golden Gate cloning strategy. (A) In order to clone the upstream (UHA) and downstream homology arms (DHA) of the homology directed repair (HDR) donor construct, perform PCR using specific primers and genomic fly DNA. In addition to the segments that anneal with the genomic DNA, the primers designed here have features that facilitate the subcloning of these fragments using the Golden Gate strategy. (B) In addition to the two homology arms generated by PCR, this protocol requires two plasmids, one that provides the vector backbone of the final product (pBH vector shown on the left) and another that provides the yellow[wing2+] cassette. (C) Assembly of the amx-yellow[wing2+] HDR plasmid through the Golden Gate reaction. By mixing the UHA and DHA from (A) , the two plasmids from (B) , a type IIs restriction enzyme BsaI and a DNA ligase, the four segments will be assembled into one plasmid through repetitive digestion and ligation reactions based on the specific overhangs created by the BsaI digestion (shown as overhangs ① to ④).

    Article Snippet: 1 ul 10X T4 ligation buffer (NEB, B0202S).

    Techniques: Construct, Clone Assay, Polymerase Chain Reaction, Subcloning, Generated, Plasmid Preparation, Ligation

    Southern blots of catalase-defective mutants. Shown are the Southern blots of Hin dIII-digested genomic DNA from the wild type, Y4Nal (lane 2), and catalase-negative mutants, Aa1393 (lane 3), Aa1394 (lane 4), and Aa1395 (lane 5). Lane 1 contains digoxigenin-labeled DNA molecular weight markers (shown in kilobases) (Boehringer Mannheim). Probes were digoxigenin-labeled DNA containing the kan gene (A) or the katA gene (B).

    Journal: Journal of Bacteriology

    Article Title: Direct Selection of IS903 Transposon Insertions by Use of a Broad-Host-Range Vector: Isolation of Catalase-Deficient Mutants of Actinobacillus actinomycetemcomitans

    doi:

    Figure Lengend Snippet: Southern blots of catalase-defective mutants. Shown are the Southern blots of Hin dIII-digested genomic DNA from the wild type, Y4Nal (lane 2), and catalase-negative mutants, Aa1393 (lane 3), Aa1394 (lane 4), and Aa1395 (lane 5). Lane 1 contains digoxigenin-labeled DNA molecular weight markers (shown in kilobases) (Boehringer Mannheim). Probes were digoxigenin-labeled DNA containing the kan gene (A) or the katA gene (B).

    Article Snippet: A 1-μg portion of Hin dIII-digested genomic DNA was ligated with 10 U of DNA ligase (New England Biolabs) in a 50-μl reaction mixture at 14°C for 16 h. The large reaction volume was used to facilitate intramolecular ligation.

    Techniques: Labeling, Molecular Weight