dna ligase buffer  (New England Biolabs)


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    Structured Review

    New England Biolabs dna ligase buffer
    The genomic map of fPS-7. The predicted genes are arranged in the direction of transcription shown by different colored arrows. Genes involved in nucleotide metabolism, <t>DNA</t> replication, recombination or repair are shown in green. Genes involved in morphogenesis and virion structures are depicted in brown. Genes involved in DNA packaging and lysis, are shown in blue and red, respectively. Genes coding for hypothetical proteins or conserved phage proteins of unknown function are shown in light grey. Homing endonucleases are shown in yellow. Direct terminal repeats (DTRs) are shown in black. On top of the genome, the host RNA polymerase (RNAP)-dependent promoters are shown with red double-arrows labelled with −35 and −10, and the phage RNAP-dependent promoters with black arrows labelled from P1 to P12. Terminators are shown along the genome as purple triangles and labelled from T1 to <t>T4.</t> The genetic map was created using the Geneious software.
    Dna Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Genomic Characterization of Sixteen Yersinia enterocolitica-Infecting Podoviruses of Pig Origin"

    Article Title: Genomic Characterization of Sixteen Yersinia enterocolitica-Infecting Podoviruses of Pig Origin

    Journal: Viruses

    doi: 10.3390/v10040174

    The genomic map of fPS-7. The predicted genes are arranged in the direction of transcription shown by different colored arrows. Genes involved in nucleotide metabolism, DNA replication, recombination or repair are shown in green. Genes involved in morphogenesis and virion structures are depicted in brown. Genes involved in DNA packaging and lysis, are shown in blue and red, respectively. Genes coding for hypothetical proteins or conserved phage proteins of unknown function are shown in light grey. Homing endonucleases are shown in yellow. Direct terminal repeats (DTRs) are shown in black. On top of the genome, the host RNA polymerase (RNAP)-dependent promoters are shown with red double-arrows labelled with −35 and −10, and the phage RNAP-dependent promoters with black arrows labelled from P1 to P12. Terminators are shown along the genome as purple triangles and labelled from T1 to T4. The genetic map was created using the Geneious software.
    Figure Legend Snippet: The genomic map of fPS-7. The predicted genes are arranged in the direction of transcription shown by different colored arrows. Genes involved in nucleotide metabolism, DNA replication, recombination or repair are shown in green. Genes involved in morphogenesis and virion structures are depicted in brown. Genes involved in DNA packaging and lysis, are shown in blue and red, respectively. Genes coding for hypothetical proteins or conserved phage proteins of unknown function are shown in light grey. Homing endonucleases are shown in yellow. Direct terminal repeats (DTRs) are shown in black. On top of the genome, the host RNA polymerase (RNAP)-dependent promoters are shown with red double-arrows labelled with −35 and −10, and the phage RNAP-dependent promoters with black arrows labelled from P1 to P12. Terminators are shown along the genome as purple triangles and labelled from T1 to T4. The genetic map was created using the Geneious software.

    Techniques Used: Lysis, Software

    2) Product Images from "Genomic Characterization of Sixteen Yersinia enterocolitica-Infecting Podoviruses of Pig Origin"

    Article Title: Genomic Characterization of Sixteen Yersinia enterocolitica-Infecting Podoviruses of Pig Origin

    Journal: Viruses

    doi: 10.3390/v10040174

    The genomic map of fPS-7. The predicted genes are arranged in the direction of transcription shown by different colored arrows. Genes involved in nucleotide metabolism, DNA replication, recombination or repair are shown in green. Genes involved in morphogenesis and virion structures are depicted in brown. Genes involved in DNA packaging and lysis, are shown in blue and red, respectively. Genes coding for hypothetical proteins or conserved phage proteins of unknown function are shown in light grey. Homing endonucleases are shown in yellow. Direct terminal repeats (DTRs) are shown in black. On top of the genome, the host RNA polymerase (RNAP)-dependent promoters are shown with red double-arrows labelled with −35 and −10, and the phage RNAP-dependent promoters with black arrows labelled from P1 to P12. Terminators are shown along the genome as purple triangles and labelled from T1 to T4. The genetic map was created using the Geneious software.
    Figure Legend Snippet: The genomic map of fPS-7. The predicted genes are arranged in the direction of transcription shown by different colored arrows. Genes involved in nucleotide metabolism, DNA replication, recombination or repair are shown in green. Genes involved in morphogenesis and virion structures are depicted in brown. Genes involved in DNA packaging and lysis, are shown in blue and red, respectively. Genes coding for hypothetical proteins or conserved phage proteins of unknown function are shown in light grey. Homing endonucleases are shown in yellow. Direct terminal repeats (DTRs) are shown in black. On top of the genome, the host RNA polymerase (RNAP)-dependent promoters are shown with red double-arrows labelled with −35 and −10, and the phage RNAP-dependent promoters with black arrows labelled from P1 to P12. Terminators are shown along the genome as purple triangles and labelled from T1 to T4. The genetic map was created using the Geneious software.

    Techniques Used: Lysis, Software

    3) Product Images from "CLT-seq as a universal homopolymer-sequencing concept reveals poly(A)-tail-tuned ncRNA regulation"

    Article Title: CLT-seq as a universal homopolymer-sequencing concept reveals poly(A)-tail-tuned ncRNA regulation

    Journal: bioRxiv

    doi: 10.1101/2022.09.11.507502

    Transcriptome-wide poly(A)-tail length determination and validation. a ) The stepwise procedure of transcriptome-wide tail-length determination by CLT-seq. b ) Mapping read coverage over 59 000 transcripts across six oligo(dT) primers based on paired-end R 1 and R 2 reads. c ) Scalar and matrix operation for poly(A) rescaling, frequency weighting, and mean poly(A)-tail length determining. T 12 (red), T 18 (cyan), or T 30 (violet) primer takes a different set of parameters. The arrow means the operating process. d ) Global profile of poly(A) tail in HEK293T with T 12 (red), T 18 (cyan), or T 30 (violet)-based CLT-seq. GAPDH-specific poly(A) tail profile from T 12 -based CLT-seq, n=2 (two replicates). e ) Poly(A) length validation by LM-PAT assay for GAPDH with two replicates. PAT assay product identified by Agilent 2100 bioanalyzer with DNA chip in left. PAT assay profile (blue) with RFU ( a.u. ) overlapped the poly(A) length distribution (red) with weighted frequency (%) restored from CLT-seq data in the right panel.
    Figure Legend Snippet: Transcriptome-wide poly(A)-tail length determination and validation. a ) The stepwise procedure of transcriptome-wide tail-length determination by CLT-seq. b ) Mapping read coverage over 59 000 transcripts across six oligo(dT) primers based on paired-end R 1 and R 2 reads. c ) Scalar and matrix operation for poly(A) rescaling, frequency weighting, and mean poly(A)-tail length determining. T 12 (red), T 18 (cyan), or T 30 (violet) primer takes a different set of parameters. The arrow means the operating process. d ) Global profile of poly(A) tail in HEK293T with T 12 (red), T 18 (cyan), or T 30 (violet)-based CLT-seq. GAPDH-specific poly(A) tail profile from T 12 -based CLT-seq, n=2 (two replicates). e ) Poly(A) length validation by LM-PAT assay for GAPDH with two replicates. PAT assay product identified by Agilent 2100 bioanalyzer with DNA chip in left. PAT assay profile (blue) with RFU ( a.u. ) overlapped the poly(A) length distribution (red) with weighted frequency (%) restored from CLT-seq data in the right panel.

    Techniques Used: Chromatin Immunoprecipitation

    4) Product Images from "High-Mobility-Group Box Nuclear Factors of Plasmodium falciparum †"

    Article Title: High-Mobility-Group Box Nuclear Factors of Plasmodium falciparum †

    Journal:

    doi: 10.1128/EC.5.4.672-682.2006

    EMSA interaction between cruciform DNA and either rePfHMGB1 (a) or rePfHMGB2 (b). Increasing concentrations (0 to 25 μM) of rePfHMGB proteins were incubated with radiolabeled 4H. Competition EMSA experiments were performed between the rePfHMGB1-4H
    Figure Legend Snippet: EMSA interaction between cruciform DNA and either rePfHMGB1 (a) or rePfHMGB2 (b). Increasing concentrations (0 to 25 μM) of rePfHMGB proteins were incubated with radiolabeled 4H. Competition EMSA experiments were performed between the rePfHMGB1-4H

    Techniques Used: Incubation

    DNA bending and ligase-mediated circularization assay with either rePfHMGB1 (a) or rePfHMGB2 (b). The γ- 32 P 5′ end-labeled 123-bp DNA fragment was preincubated with increasing amounts of rePfHMGB1 (0 to 2 μM) or rePfHMGB2 (0 to
    Figure Legend Snippet: DNA bending and ligase-mediated circularization assay with either rePfHMGB1 (a) or rePfHMGB2 (b). The γ- 32 P 5′ end-labeled 123-bp DNA fragment was preincubated with increasing amounts of rePfHMGB1 (0 to 2 μM) or rePfHMGB2 (0 to

    Techniques Used: Labeling

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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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    Average 97 stars, based on 1 article reviews
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    New England Biolabs t4 dna ligase buffer
    Structure-seq2 leads to a lower ligation bias. ( A ) After RT (Figure 1 , step 1A/1B), excess of the 27 nt primer (blue, top, right) is still present in the solution. During ligation (Figure 1 , step 3A/3B), this primer can also ligate to the 40 nt hairpin adaptor (pink) to form an unwanted 67 nt by-product which has no insert and so results in sequencing reads with no utility. ( B ) The complement of the first nucleotide after the adaptor sequence read during sequencing is the nucleotide that ligated to the adaptor. Our new <t>T4</t> DNA ligase-based method (green, –DMS and pink, +DMS) substantially decreases ligation bias as compared to the previous Circligase-based method (blue). Percentages equaling the transcriptomic distribution of the four nucleotides (black) are ideal.
    T4 Dna Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase buffer/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase buffer - by Bioz Stars, 2022-10
    99/100 stars
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    Image Search Results


    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

    Structure-seq2 leads to a lower ligation bias. ( A ) After RT (Figure 1 , step 1A/1B), excess of the 27 nt primer (blue, top, right) is still present in the solution. During ligation (Figure 1 , step 3A/3B), this primer can also ligate to the 40 nt hairpin adaptor (pink) to form an unwanted 67 nt by-product which has no insert and so results in sequencing reads with no utility. ( B ) The complement of the first nucleotide after the adaptor sequence read during sequencing is the nucleotide that ligated to the adaptor. Our new T4 DNA ligase-based method (green, –DMS and pink, +DMS) substantially decreases ligation bias as compared to the previous Circligase-based method (blue). Percentages equaling the transcriptomic distribution of the four nucleotides (black) are ideal.

    Journal: Nucleic Acids Research

    Article Title: Structure-seq2: sensitive and accurate genome-wide profiling of RNA structure in vivo

    doi: 10.1093/nar/gkx533

    Figure Lengend Snippet: Structure-seq2 leads to a lower ligation bias. ( A ) After RT (Figure 1 , step 1A/1B), excess of the 27 nt primer (blue, top, right) is still present in the solution. During ligation (Figure 1 , step 3A/3B), this primer can also ligate to the 40 nt hairpin adaptor (pink) to form an unwanted 67 nt by-product which has no insert and so results in sequencing reads with no utility. ( B ) The complement of the first nucleotide after the adaptor sequence read during sequencing is the nucleotide that ligated to the adaptor. Our new T4 DNA ligase-based method (green, –DMS and pink, +DMS) substantially decreases ligation bias as compared to the previous Circligase-based method (blue). Percentages equaling the transcriptomic distribution of the four nucleotides (black) are ideal.

    Article Snippet: After renaturing the purified cDNA with betaine, polyethylene glycol 8000 (PEG 8000) and hairpin donor (5′-pTGAAGAGCCTAGTCGCTGTTCANNNNNNCTGCCCATAGAG-3′-Spacer, where ‘5′-p’ is a 5′ phosphate and ‘3′-Spacer’ is a 3-carbon linker), 10× T4 DNA ligase buffer and T4 DNA ligase (NEB) were added to give a final 10 μl reaction mixture containing 500 mM Betaine, 20% PEG 8000, 10 μM hairpin donor, 1× T4 DNA ligase buffer, and 400 U T4 DNA ligase.

    Techniques: Ligation, Sequencing

    Two versions of Structure-seq2 produce high quality data. In Structure-seq2, RNA (kelly green) is first modified by DMS or another chemical that can be read-out through reverse transcription. The RNA is then prepared for Illumina NGS sequencing by conversion to cDNA (Step 1A/1B, blue), ligating an adaptor (Step 3A/3B), and amplifying the products while incorporating TruSeq primer sequences (Step 5A/5B). In order to increase library quality, numerous improvements were made to the original Structure-seq protocol (boxed). These include performing the ligation with a hairpin adaptor and T4 DNA ligase (Step 3A/3B; pink) ( 10 ), and adding various purification steps to remove a deleterious by-product (Figure 2A ). We present two options for purification: PAGE purification ( A ) or a biotin–streptavidin pull down ( B ). In the PAGE purification method, an additional PAGE purification step is added after reverse transcription (Step 2A). In the biotin–streptavidin pull down method, biotinylated dNTPs (cyan) are incorporated into the extended product during reverse transcription (Step 1B) and are purified via a magnetic streptavidin pull down after reverse transcription (Step 2B) and after ligation (Step 4B). There is also a common, final PAGE purification step following amplification (Step 5A/5B). Finally, a custom sequencing primer (light green) is used during sequencing (Step 7A/7B) to further provide high quality data. Supplementary Figure S1 is a version of this figure with all the nucleotides shown explicitly.

    Journal: Nucleic Acids Research

    Article Title: Structure-seq2: sensitive and accurate genome-wide profiling of RNA structure in vivo

    doi: 10.1093/nar/gkx533

    Figure Lengend Snippet: Two versions of Structure-seq2 produce high quality data. In Structure-seq2, RNA (kelly green) is first modified by DMS or another chemical that can be read-out through reverse transcription. The RNA is then prepared for Illumina NGS sequencing by conversion to cDNA (Step 1A/1B, blue), ligating an adaptor (Step 3A/3B), and amplifying the products while incorporating TruSeq primer sequences (Step 5A/5B). In order to increase library quality, numerous improvements were made to the original Structure-seq protocol (boxed). These include performing the ligation with a hairpin adaptor and T4 DNA ligase (Step 3A/3B; pink) ( 10 ), and adding various purification steps to remove a deleterious by-product (Figure 2A ). We present two options for purification: PAGE purification ( A ) or a biotin–streptavidin pull down ( B ). In the PAGE purification method, an additional PAGE purification step is added after reverse transcription (Step 2A). In the biotin–streptavidin pull down method, biotinylated dNTPs (cyan) are incorporated into the extended product during reverse transcription (Step 1B) and are purified via a magnetic streptavidin pull down after reverse transcription (Step 2B) and after ligation (Step 4B). There is also a common, final PAGE purification step following amplification (Step 5A/5B). Finally, a custom sequencing primer (light green) is used during sequencing (Step 7A/7B) to further provide high quality data. Supplementary Figure S1 is a version of this figure with all the nucleotides shown explicitly.

    Article Snippet: After renaturing the purified cDNA with betaine, polyethylene glycol 8000 (PEG 8000) and hairpin donor (5′-pTGAAGAGCCTAGTCGCTGTTCANNNNNNCTGCCCATAGAG-3′-Spacer, where ‘5′-p’ is a 5′ phosphate and ‘3′-Spacer’ is a 3-carbon linker), 10× T4 DNA ligase buffer and T4 DNA ligase (NEB) were added to give a final 10 μl reaction mixture containing 500 mM Betaine, 20% PEG 8000, 10 μM hairpin donor, 1× T4 DNA ligase buffer, and 400 U T4 DNA ligase.

    Techniques: Modification, Next-Generation Sequencing, Sequencing, Ligation, Purification, Polyacrylamide Gel Electrophoresis, Amplification