t4 dna ligase ligation protocol  (New England Biolabs)


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    Name:
    T4 DNA Ligase
    Description:
    T4 DNA Ligase 100 000 units
    Catalog Number:
    m0202l
    Price:
    256
    Size:
    100 000 units
    Category:
    DNA Ligases
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    Structured Review

    New England Biolabs t4 dna ligase ligation protocol
    T4 DNA Ligase
    T4 DNA Ligase 100 000 units
    https://www.bioz.com/result/t4 dna ligase ligation protocol/product/New England Biolabs
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase ligation protocol - by Bioz Stars, 2020-01
    90/100 stars

    Related Products / Commonly Used Together

    dsdna inhibition likely
    standard sticky-end ligation

    Images

    1) Product Images from "The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase"

    Article Title: The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0150802

    Effect of Inhibiting dsDNA on Enzyme Self-Adenylylation Rate. The determined rates for self-adenylylation of an uninhibited reaction, 2.5 μM T4 DNA ligase (red) and 2.5 μM T4 DNA ligase and inhibited reactions 2.5 μM DNA (blue) and 10 μM DNA (green). The reactions were fit to a single exponential equation ( Eq 6 ) to determine the reaction rate. The uninhibited reaction was determined to have a single turnover rate of 20 s -1 ± 2 s -1 . While the 2.5 μM inhibited reaction had a single turnover rate of 2.8 s -1 ± 0.5 s -1 and the 10 μM inhibited reaction had a single turnover rate of 1.0 s -1 ± 1 s -1 . All reactions were performed a minimum of three times at 16°C. Error reported is the standard error for the replicates.
    Figure Legend Snippet: Effect of Inhibiting dsDNA on Enzyme Self-Adenylylation Rate. The determined rates for self-adenylylation of an uninhibited reaction, 2.5 μM T4 DNA ligase (red) and 2.5 μM T4 DNA ligase and inhibited reactions 2.5 μM DNA (blue) and 10 μM DNA (green). The reactions were fit to a single exponential equation ( Eq 6 ) to determine the reaction rate. The uninhibited reaction was determined to have a single turnover rate of 20 s -1 ± 2 s -1 . While the 2.5 μM inhibited reaction had a single turnover rate of 2.8 s -1 ± 0.5 s -1 and the 10 μM inhibited reaction had a single turnover rate of 1.0 s -1 ± 1 s -1 . All reactions were performed a minimum of three times at 16°C. Error reported is the standard error for the replicates.

    Techniques Used:

    T4 DNA Ligase Reaction Model. Modified reaction pathway to include the newly observed reactions in the previously described DNA ligation pathway that are inhibited by the presence of non-nicked dsDNA. A . Non-nicked dsDNA can bind to the deadenylylated form of the enzyme inhibition formation of the adenylylated form of the enzyme. B . Non-nicked dsDNA binds to the Lig-AMP form, preventing complexation with its preferred ds-nDNA substrate.
    Figure Legend Snippet: T4 DNA Ligase Reaction Model. Modified reaction pathway to include the newly observed reactions in the previously described DNA ligation pathway that are inhibited by the presence of non-nicked dsDNA. A . Non-nicked dsDNA can bind to the deadenylylated form of the enzyme inhibition formation of the adenylylated form of the enzyme. B . Non-nicked dsDNA binds to the Lig-AMP form, preventing complexation with its preferred ds-nDNA substrate.

    Techniques Used: Modification, DNA Ligation, Enzyme Inhibition Assay

    k cat /K m Curve for T4 DNA Ligase. The data was obtained through titration of increasing concentrations of a 75mer-ds-nDNA substrate, reacted at 16°C to determine initial reaction rates. T4 DNA ligase concentrations used were 25 pM– 100 pM. The initial rates were plotted against their respective substrate concentrations and fit by: A . a classical uncompetitive substrate inhibition model ( Eq 2 ), where k cat and K m Values of 0.44 s -1 ± 0.3 s -1 and 4 nM ± 1 nM respectively, were determined. The K i value for substrate inhibition was calculated to be 590 nM ± 170 nM. B . A competitive substrate inhibition for a Bi-Bi Ping-Pong mechanism ( Eq 3 ) k cat and K m values of 0.48 s -1 ± 0.3 s -1 and 4 nM ± 1 nM respectively, were determined. The K i value for substrate inhibition was calculated to be 54 nM ± 15 nM. All data points are the average of at least three independent experiments, and the error reported is the standard deviation for the replicates.
    Figure Legend Snippet: k cat /K m Curve for T4 DNA Ligase. The data was obtained through titration of increasing concentrations of a 75mer-ds-nDNA substrate, reacted at 16°C to determine initial reaction rates. T4 DNA ligase concentrations used were 25 pM– 100 pM. The initial rates were plotted against their respective substrate concentrations and fit by: A . a classical uncompetitive substrate inhibition model ( Eq 2 ), where k cat and K m Values of 0.44 s -1 ± 0.3 s -1 and 4 nM ± 1 nM respectively, were determined. The K i value for substrate inhibition was calculated to be 590 nM ± 170 nM. B . A competitive substrate inhibition for a Bi-Bi Ping-Pong mechanism ( Eq 3 ) k cat and K m values of 0.48 s -1 ± 0.3 s -1 and 4 nM ± 1 nM respectively, were determined. The K i value for substrate inhibition was calculated to be 54 nM ± 15 nM. All data points are the average of at least three independent experiments, and the error reported is the standard deviation for the replicates.

    Techniques Used: Titration, Inhibition, Standard Deviation

    Various Inhibitors Effects on Rate of Nick Sealing. Various concentrations of dsDNA substrates were utilized as potential inhibitors of the T4 DNA ligase steady state ligation reaction on 20 nM of the 75mer-ds-nDNA substrate. All reactions were performed in the presence of 25 pM of T4 DNA ligase, a minimum of three times at 16°C. Error reported is the standard deviation for the replicates.
    Figure Legend Snippet: Various Inhibitors Effects on Rate of Nick Sealing. Various concentrations of dsDNA substrates were utilized as potential inhibitors of the T4 DNA ligase steady state ligation reaction on 20 nM of the 75mer-ds-nDNA substrate. All reactions were performed in the presence of 25 pM of T4 DNA ligase, a minimum of three times at 16°C. Error reported is the standard deviation for the replicates.

    Techniques Used: Ligation, Standard Deviation

    Competition for ds-nDNA-Binding by dsDNA. Lane one contains 4 nM of the 75mer-ds-nDNA substrate alone, lanes 2–6 show shifting of the 4 nM substrate into a completely bound state as the concentration of T4 DNA ligase is increased from 100 nM– 1000 nM. Lanes 7–11 are of a titration of increasing concentrations of the unlabeled I-75-dsDNA oligo into a reaction containing 4 nM labeled nicked substrate and 1000 nM T4 DNA ligase. EMSA reactions were all performed and electrophoresed at room temperature (22°C).
    Figure Legend Snippet: Competition for ds-nDNA-Binding by dsDNA. Lane one contains 4 nM of the 75mer-ds-nDNA substrate alone, lanes 2–6 show shifting of the 4 nM substrate into a completely bound state as the concentration of T4 DNA ligase is increased from 100 nM– 1000 nM. Lanes 7–11 are of a titration of increasing concentrations of the unlabeled I-75-dsDNA oligo into a reaction containing 4 nM labeled nicked substrate and 1000 nM T4 DNA ligase. EMSA reactions were all performed and electrophoresed at room temperature (22°C).

    Techniques Used: Binding Assay, Concentration Assay, Titration, Labeling

    Related Articles

    Clone Assay:

    Article Title: SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome
    Article Snippet: Cloned pCDNATM 3.1 (−) expression vectors were also PCR amplified with unique primers harbouring specific modifications to allow the transfer of wild-type and mutant inserts into pEGFP-N2 constructs (adding EcoRI and BamHI restriction sites with poly adenine for optimized restriction while abrogating the FLAG sequence and stop codon and maintaining reading frame). .. Ligations were done using T4 DNA ligase (New England Biolabs).

    Article Title: Silencing of Rieske Iron-Sulfur Protein Impacts Upon the Development and Reproduction of Spodoptera exigua by Regulating ATP Synthesis
    Article Snippet: For each insect, the open reading flame (ORF) of RISPs was cloned from the appropriate cDNA by using a specific set of primers: SeRISPOF, SeRISPOR, SlRISPOF, SlRISPOR, PxRISPOF, PxRISPOR, BmRISPOF, and BmRISPOR (Table ). .. Then, 50 μL digestion reactions were set up consisting of 2 μg of DNA, 5 μL of 10 × NEB buffer 4, 1 μL of 100 × BSA and 1 μL of 20 U/μL enzyme; these were incubated at 37°C for 4 h. Digested RISPs and pET32a expression vectors were then purified using a Universal DNA Purification Kit (TIANGEN) in accordance with the manufacturer's recommendations and then ligated together using T4 DNA Ligase (New England BioLabs).

    Article Title: CRISPR-FRT targets shared sites in a knock-out collection for off-the-shelf genome editing
    Article Snippet: For cloning the FRT-gRNA into pTargetF, we performed an inverse-PCR type reaction applied to the whole plasmid using oligonucleotide primers gRNA:FRT and gRNA:LexA-G85-2 (Supplementary Data ). .. However, in the latter case the resulting PCR product was cut with SpeI (NEB), the ends self-ligated with T4 DNA ligase (New England Biolabs), phenol:chloroform extracted, ethanol precipitated and transformed into DH5α cells.

    Amplification:

    Article Title: SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome
    Article Snippet: Cloned pCDNATM 3.1 (−) expression vectors were also PCR amplified with unique primers harbouring specific modifications to allow the transfer of wild-type and mutant inserts into pEGFP-N2 constructs (adding EcoRI and BamHI restriction sites with poly adenine for optimized restriction while abrogating the FLAG sequence and stop codon and maintaining reading frame). .. Ligations were done using T4 DNA ligase (New England Biolabs).

    Article Title: FdC1 and Leaf-Type Ferredoxins Channel Electrons From Photosystem I to Different Downstream Electron Acceptors
    Article Snippet: The coding sequences of the candidate photosynthetic and non-photosynthetic apparatus subunits were amplified from the cDNA template using specific primers ( Supplementary Table ) by Pfx polymerase (Invitrogen, United States) and KAPA HiFiTM HotStart ReadyMix (Kapa Biosystems, United States). .. The restricted PCR products were ligated into corresponding vectors by the T4 DNA ligase (NEB, United States).

    Article Title: Crystal structure of intraflagellar transport protein 80 reveals a homo-dimer required for ciliogenesis
    Article Snippet: Ligations were carried out in 10 µl reactions in 1X T4 DNA ligase buffer (NEB), using 50 ng linear pX330, 1 µl annealed oligos (0.5 µM) and 1 µl T4 DNA ligase (NEB), and were incubated at 4°C for 16 hr. .. Expression vectors were amplified and purified by Plasmid Midi Kit (QIAGEN, Germany).

    Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals
    Article Snippet: The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; M0202) ligation protocol. .. The pWPT-ΔGFP-X plasmids encoding the native form of the X proteins were obtained after digestion of the pWPT-GFP-X vectors using MluI and NotI and amplification of each X using Mlu1-pWPT-X-F primer (5′-GCT TAC GCG TTC TGC AGT CGA CGA ATT CAC CAT G-3′) and pWPT-R primer (5′-GTC AGC AAA CAC AGT GCA CAC CA-3′).

    Binding Assay:

    Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies
    Article Snippet: Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U T4 DNA ligase NEB M0202S, final sample volume 1.2 ml) and samples were incubated for 4 h at room temperature under rotation. .. Biotinylated Hi-C DNA in 1X binding buffer (5 mM Tris-HCl, pH 8, 0.5 mM EDTA, 1 M NaCl) was pulled down using Dynabeads MyOne Streptavidin C1 (Life Technologies, 650.01), using 5 µl of beads per microgram of DNA, pre-washed in 1X binding buffer.

    Synthesized:

    Article Title: Silencing of Rieske Iron-Sulfur Protein Impacts Upon the Development and Reproduction of Spodoptera exigua by Regulating ATP Synthesis
    Article Snippet: Total RNA from the third instar larva of S. exigua, S. litura, P. xylostella , and B. mori were isolated and the cDNAs synthesized as described above. .. Then, 50 μL digestion reactions were set up consisting of 2 μg of DNA, 5 μL of 10 × NEB buffer 4, 1 μL of 100 × BSA and 1 μL of 20 U/μL enzyme; these were incubated at 37°C for 4 h. Digested RISPs and pET32a expression vectors were then purified using a Universal DNA Purification Kit (TIANGEN) in accordance with the manufacturer's recommendations and then ligated together using T4 DNA Ligase (New England BioLabs).

    Article Title: FdC1 and Leaf-Type Ferredoxins Channel Electrons From Photosystem I to Different Downstream Electron Acceptors
    Article Snippet: A first-strand cDNA library was synthesized by M-MLV reverse transcription (Promega, United States) with oligo dT15 primers and was then used as a template for the polymerase chain reaction (PCR). .. The restricted PCR products were ligated into corresponding vectors by the T4 DNA ligase (NEB, United States).

    Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals
    Article Snippet: The X coding regions (synthesized by Genewiz) from hepadnaviruses infecting the New World wooly monkey ( Lagothrix ) (WMHBx) and three distant bat species, including the roundleaf bat ( Hipposideros cf. ruber ), the horseshoe bat ( Rhinolophus Alcyone ), and the tent-making bat ( Uroderma bilobatum ) (RBHBx, HBHBx, TBHBx, respectively) , were expressed from the same vector. .. The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; M0202) ligation protocol.

    Construct:

    Article Title: SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome
    Article Snippet: Paragraph title: SLC30A9 (ZnT-9) constructs ... Ligations were done using T4 DNA ligase (New England Biolabs).

    cDNA Library Assay:

    Article Title: FdC1 and Leaf-Type Ferredoxins Channel Electrons From Photosystem I to Different Downstream Electron Acceptors
    Article Snippet: A first-strand cDNA library was synthesized by M-MLV reverse transcription (Promega, United States) with oligo dT15 primers and was then used as a template for the polymerase chain reaction (PCR). .. The restricted PCR products were ligated into corresponding vectors by the T4 DNA ligase (NEB, United States).

    Incubation:

    Article Title: Silencing of Rieske Iron-Sulfur Protein Impacts Upon the Development and Reproduction of Spodoptera exigua by Regulating ATP Synthesis
    Article Snippet: .. Then, 50 μL digestion reactions were set up consisting of 2 μg of DNA, 5 μL of 10 × NEB buffer 4, 1 μL of 100 × BSA and 1 μL of 20 U/μL enzyme; these were incubated at 37°C for 4 h. Digested RISPs and pET32a expression vectors were then purified using a Universal DNA Purification Kit (TIANGEN) in accordance with the manufacturer's recommendations and then ligated together using T4 DNA Ligase (New England BioLabs). .. The 10 μL ligation reaction consisted of 5 μL of purified RISPs, 3 μL of purified vector, 1 μL of 10 × T4 DNA ligase buffer, and 1 μL of T4 DNA ligase.

    Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies
    Article Snippet: .. Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U T4 DNA ligase NEB M0202S, final sample volume 1.2 ml) and samples were incubated for 4 h at room temperature under rotation. ..

    Article Title: Crystal structure of intraflagellar transport protein 80 reveals a homo-dimer required for ciliogenesis
    Article Snippet: .. Ligations were carried out in 10 µl reactions in 1X T4 DNA ligase buffer (NEB), using 50 ng linear pX330, 1 µl annealed oligos (0.5 µM) and 1 µl T4 DNA ligase (NEB), and were incubated at 4°C for 16 hr. .. Correct sgRNA insertion was confirmed by sequencing.

    Expressing:

    Article Title: SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome
    Article Snippet: PCR products were then restricted using EcoRI and BamHI enzymes and ligated into pEGFP-N2 expression vector. .. Ligations were done using T4 DNA ligase (New England Biolabs).

    Article Title: Silencing of Rieske Iron-Sulfur Protein Impacts Upon the Development and Reproduction of Spodoptera exigua by Regulating ATP Synthesis
    Article Snippet: .. Then, 50 μL digestion reactions were set up consisting of 2 μg of DNA, 5 μL of 10 × NEB buffer 4, 1 μL of 100 × BSA and 1 μL of 20 U/μL enzyme; these were incubated at 37°C for 4 h. Digested RISPs and pET32a expression vectors were then purified using a Universal DNA Purification Kit (TIANGEN) in accordance with the manufacturer's recommendations and then ligated together using T4 DNA Ligase (New England BioLabs). .. The 10 μL ligation reaction consisted of 5 μL of purified RISPs, 3 μL of purified vector, 1 μL of 10 × T4 DNA ligase buffer, and 1 μL of T4 DNA ligase.

    Article Title: Crystal structure of intraflagellar transport protein 80 reveals a homo-dimer required for ciliogenesis
    Article Snippet: To make respective CRISPR expression vectors, gRNAs annealed oligos were ligated in linearised pX330. .. Ligations were carried out in 10 µl reactions in 1X T4 DNA ligase buffer (NEB), using 50 ng linear pX330, 1 µl annealed oligos (0.5 µM) and 1 µl T4 DNA ligase (NEB), and were incubated at 4°C for 16 hr.

    Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals
    Article Snippet: Paragraph title: Expression plasmids. ... The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; M0202) ligation protocol.

    Western Blot:

    Article Title: Silencing of Rieske Iron-Sulfur Protein Impacts Upon the Development and Reproduction of Spodoptera exigua by Regulating ATP Synthesis
    Article Snippet: Paragraph title: Prokaryotic expression of recombinant protein and western-blotting ... Then, 50 μL digestion reactions were set up consisting of 2 μg of DNA, 5 μL of 10 × NEB buffer 4, 1 μL of 100 × BSA and 1 μL of 20 U/μL enzyme; these were incubated at 37°C for 4 h. Digested RISPs and pET32a expression vectors were then purified using a Universal DNA Purification Kit (TIANGEN) in accordance with the manufacturer's recommendations and then ligated together using T4 DNA Ligase (New England BioLabs).

    Transformation Assay:

    Article Title: Silencing of Rieske Iron-Sulfur Protein Impacts Upon the Development and Reproduction of Spodoptera exigua by Regulating ATP Synthesis
    Article Snippet: Then, 50 μL digestion reactions were set up consisting of 2 μg of DNA, 5 μL of 10 × NEB buffer 4, 1 μL of 100 × BSA and 1 μL of 20 U/μL enzyme; these were incubated at 37°C for 4 h. Digested RISPs and pET32a expression vectors were then purified using a Universal DNA Purification Kit (TIANGEN) in accordance with the manufacturer's recommendations and then ligated together using T4 DNA Ligase (New England BioLabs). .. Then, the reaction tubes were incubated at 16°C for 12 h. Recombinant plasmids (pET32a-RISPs) were then transformed into Escherichia coli Transetta (DE3), using a non-carrier pET32a vector as a negative control.

    Article Title: CRISPR-FRT targets shared sites in a knock-out collection for off-the-shelf genome editing
    Article Snippet: .. However, in the latter case the resulting PCR product was cut with SpeI (NEB), the ends self-ligated with T4 DNA ligase (New England Biolabs), phenol:chloroform extracted, ethanol precipitated and transformed into DH5α cells. ..

    Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals
    Article Snippet: The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; M0202) ligation protocol. .. The plasmid was then transformed following the high-efficiency transformation protocol using NEB 10-beta Competent Escherichia coli (NEB; C3019).

    Transfection:

    Article Title: Crystal structure of intraflagellar transport protein 80 reveals a homo-dimer required for ciliogenesis
    Article Snippet: Ligations were carried out in 10 µl reactions in 1X T4 DNA ligase buffer (NEB), using 50 ng linear pX330, 1 µl annealed oligos (0.5 µM) and 1 µl T4 DNA ligase (NEB), and were incubated at 4°C for 16 hr. .. In a well of a six well plate, 40–60% confluent IMCD3 cells were transfected with 2.4 µg plasmid DNA, using 8.5 µl Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) in 300 µl Opti-MEM reduced serum medium (Thermo Fisher Scientific).

    Inverse PCR:

    Article Title: CRISPR-FRT targets shared sites in a knock-out collection for off-the-shelf genome editing
    Article Snippet: For cloning the FRT-gRNA into pTargetF, we performed an inverse-PCR type reaction applied to the whole plasmid using oligonucleotide primers gRNA:FRT and gRNA:LexA-G85-2 (Supplementary Data ). .. However, in the latter case the resulting PCR product was cut with SpeI (NEB), the ends self-ligated with T4 DNA ligase (New England Biolabs), phenol:chloroform extracted, ethanol precipitated and transformed into DH5α cells.

    Ligation:

    Article Title: Silencing of Rieske Iron-Sulfur Protein Impacts Upon the Development and Reproduction of Spodoptera exigua by Regulating ATP Synthesis
    Article Snippet: Then, 50 μL digestion reactions were set up consisting of 2 μg of DNA, 5 μL of 10 × NEB buffer 4, 1 μL of 100 × BSA and 1 μL of 20 U/μL enzyme; these were incubated at 37°C for 4 h. Digested RISPs and pET32a expression vectors were then purified using a Universal DNA Purification Kit (TIANGEN) in accordance with the manufacturer's recommendations and then ligated together using T4 DNA Ligase (New England BioLabs). .. The 10 μL ligation reaction consisted of 5 μL of purified RISPs, 3 μL of purified vector, 1 μL of 10 × T4 DNA ligase buffer, and 1 μL of T4 DNA ligase.

    Article Title: CRISPR-FRT targets shared sites in a knock-out collection for off-the-shelf genome editing
    Article Snippet: Next, we used ligation-independent cloning or Gibson assembly (New England Biolabs, NEB) to include the FRT protospacer sequence into the pKDsgRNA gRNA delivery plasmid. .. However, in the latter case the resulting PCR product was cut with SpeI (NEB), the ends self-ligated with T4 DNA ligase (New England Biolabs), phenol:chloroform extracted, ethanol precipitated and transformed into DH5α cells.

    Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies
    Article Snippet: .. Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U T4 DNA ligase NEB M0202S, final sample volume 1.2 ml) and samples were incubated for 4 h at room temperature under rotation. ..

    Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals
    Article Snippet: .. The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; M0202) ligation protocol. .. The plasmid was then transformed following the high-efficiency transformation protocol using NEB 10-beta Competent Escherichia coli (NEB; C3019).

    DNA Sequencing:

    Article Title: Biosynthesis of thiocarboxylic acid-containing natural products
    Article Snippet: Q5 high-fidelity DNA polymerase, restriction endonucleases, and T4 DNA ligase were purchased from NEB and used by following the protocols provided by the manufacturers. .. Q5 high-fidelity DNA polymerase, restriction endonucleases, and T4 DNA ligase were purchased from NEB and used by following the protocols provided by the manufacturers.

    Sequencing:

    Article Title: SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome
    Article Snippet: Cloned pCDNATM 3.1 (−) expression vectors were also PCR amplified with unique primers harbouring specific modifications to allow the transfer of wild-type and mutant inserts into pEGFP-N2 constructs (adding EcoRI and BamHI restriction sites with poly adenine for optimized restriction while abrogating the FLAG sequence and stop codon and maintaining reading frame). .. Ligations were done using T4 DNA ligase (New England Biolabs).

    Article Title: Silencing of Rieske Iron-Sulfur Protein Impacts Upon the Development and Reproduction of Spodoptera exigua by Regulating ATP Synthesis
    Article Snippet: Then, 50 μL digestion reactions were set up consisting of 2 μg of DNA, 5 μL of 10 × NEB buffer 4, 1 μL of 100 × BSA and 1 μL of 20 U/μL enzyme; these were incubated at 37°C for 4 h. Digested RISPs and pET32a expression vectors were then purified using a Universal DNA Purification Kit (TIANGEN) in accordance with the manufacturer's recommendations and then ligated together using T4 DNA Ligase (New England BioLabs). .. Positive recombinant E. coli were sequenced and recombinant plasmids extracted using a TIANprep Mini Plasmid Kit (TIANGEN) in order to verify sequence identity and successful ligation.

    Article Title: CRISPR-FRT targets shared sites in a knock-out collection for off-the-shelf genome editing
    Article Snippet: Next, we used ligation-independent cloning or Gibson assembly (New England Biolabs, NEB) to include the FRT protospacer sequence into the pKDsgRNA gRNA delivery plasmid. .. However, in the latter case the resulting PCR product was cut with SpeI (NEB), the ends self-ligated with T4 DNA ligase (New England Biolabs), phenol:chloroform extracted, ethanol precipitated and transformed into DH5α cells.

    Article Title: Crystal structure of intraflagellar transport protein 80 reveals a homo-dimer required for ciliogenesis
    Article Snippet: Ligations were carried out in 10 µl reactions in 1X T4 DNA ligase buffer (NEB), using 50 ng linear pX330, 1 µl annealed oligos (0.5 µM) and 1 µl T4 DNA ligase (NEB), and were incubated at 4°C for 16 hr. .. Correct sgRNA insertion was confirmed by sequencing.

    Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals
    Article Snippet: The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; M0202) ligation protocol. .. All the X-expressing plasmids were checked by Sanger sequencing using eGFP-F primer, 5′-CAT GGT CCT GCT GGA GTT CGT G-3′, and pWPT-R, 5′-GTC AGC AAA CAC AGT GCA CAC CA-3′.

    Recombinant:

    Article Title: Silencing of Rieske Iron-Sulfur Protein Impacts Upon the Development and Reproduction of Spodoptera exigua by Regulating ATP Synthesis
    Article Snippet: Paragraph title: Prokaryotic expression of recombinant protein and western-blotting ... Then, 50 μL digestion reactions were set up consisting of 2 μg of DNA, 5 μL of 10 × NEB buffer 4, 1 μL of 100 × BSA and 1 μL of 20 U/μL enzyme; these were incubated at 37°C for 4 h. Digested RISPs and pET32a expression vectors were then purified using a Universal DNA Purification Kit (TIANGEN) in accordance with the manufacturer's recommendations and then ligated together using T4 DNA Ligase (New England BioLabs).

    Hi-C:

    Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies
    Article Snippet: Paragraph title: In situ Hi-C of knockdown and control cells ... Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U T4 DNA ligase NEB M0202S, final sample volume 1.2 ml) and samples were incubated for 4 h at room temperature under rotation.

    Nucleic Acid Electrophoresis:

    Article Title: Time-Restricted PiggyBac DNA Transposition by Transposase Protein Delivery Using Lentivirus-Derived Nanoparticles
    Article Snippet: Purified DNA was ligated overnight with T4 DNA ligase (New England Biolabs) in a total volume of 500 μL. .. The products of the nested PCR were separated by gel electrophoresis, and discrete bands were excised and column purified (Gel Extraction Kit; Omega Bio-tek).

    In Vivo:

    Article Title: Silencing of Rieske Iron-Sulfur Protein Impacts Upon the Development and Reproduction of Spodoptera exigua by Regulating ATP Synthesis
    Article Snippet: Prokaryotic expression of recombinant protein and western-blotting To verify the immunocompetence between homologous RISP proteins and PxRISP antibody to, in vivo and prokaryotic protein of RISPs from S. exigua, S. litura, P. xylostella , and B. mori were exposed to PxRISP via Western-blotting. .. Then, 50 μL digestion reactions were set up consisting of 2 μg of DNA, 5 μL of 10 × NEB buffer 4, 1 μL of 100 × BSA and 1 μL of 20 U/μL enzyme; these were incubated at 37°C for 4 h. Digested RISPs and pET32a expression vectors were then purified using a Universal DNA Purification Kit (TIANGEN) in accordance with the manufacturer's recommendations and then ligated together using T4 DNA Ligase (New England BioLabs).

    Mutagenesis:

    Article Title: SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome
    Article Snippet: Cloned pCDNATM 3.1 (−) expression vectors were also PCR amplified with unique primers harbouring specific modifications to allow the transfer of wild-type and mutant inserts into pEGFP-N2 constructs (adding EcoRI and BamHI restriction sites with poly adenine for optimized restriction while abrogating the FLAG sequence and stop codon and maintaining reading frame). .. Ligations were done using T4 DNA ligase (New England Biolabs).

    Isolation:

    Article Title: Silencing of Rieske Iron-Sulfur Protein Impacts Upon the Development and Reproduction of Spodoptera exigua by Regulating ATP Synthesis
    Article Snippet: Total RNA from the third instar larva of S. exigua, S. litura, P. xylostella , and B. mori were isolated and the cDNAs synthesized as described above. .. Then, 50 μL digestion reactions were set up consisting of 2 μg of DNA, 5 μL of 10 × NEB buffer 4, 1 μL of 100 × BSA and 1 μL of 20 U/μL enzyme; these were incubated at 37°C for 4 h. Digested RISPs and pET32a expression vectors were then purified using a Universal DNA Purification Kit (TIANGEN) in accordance with the manufacturer's recommendations and then ligated together using T4 DNA Ligase (New England BioLabs).

    Article Title: Translation efficiency of heterologous proteins is significantly affected by the genetic context of RBS sequences in engineered cyanobacterium Synechocystis sp. PCC 6803
    Article Snippet: Enzymes and reagents The restriction enzymes, T4 DNA ligase and DNA polymerase used in this work were purchased from New England BioLabs (USA) or from ThermoFischer-Scientific (USA). .. Commercial Qiagen (Germany) kits were used for plasmid isolation (QIAprep Spin miniprep kit) and gel extraction (QIAquick, gel extraction kit).

    Subcloning:

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: 28704) XL1-Blue subcloning grade competent cells (Agilent, cat.no. .. M0202L) 10× T4 DNA ligase Buffer (New England BioLabs), supplied with T4 DNA ligase LB agar (Miller powder) (Fisher Scientific, cat.no.

    Purification:

    Article Title: Silencing of Rieske Iron-Sulfur Protein Impacts Upon the Development and Reproduction of Spodoptera exigua by Regulating ATP Synthesis
    Article Snippet: .. Then, 50 μL digestion reactions were set up consisting of 2 μg of DNA, 5 μL of 10 × NEB buffer 4, 1 μL of 100 × BSA and 1 μL of 20 U/μL enzyme; these were incubated at 37°C for 4 h. Digested RISPs and pET32a expression vectors were then purified using a Universal DNA Purification Kit (TIANGEN) in accordance with the manufacturer's recommendations and then ligated together using T4 DNA Ligase (New England BioLabs). .. The 10 μL ligation reaction consisted of 5 μL of purified RISPs, 3 μL of purified vector, 1 μL of 10 × T4 DNA ligase buffer, and 1 μL of T4 DNA ligase.

    Article Title: Crystal structure of intraflagellar transport protein 80 reveals a homo-dimer required for ciliogenesis
    Article Snippet: Ligations were carried out in 10 µl reactions in 1X T4 DNA ligase buffer (NEB), using 50 ng linear pX330, 1 µl annealed oligos (0.5 µM) and 1 µl T4 DNA ligase (NEB), and were incubated at 4°C for 16 hr. .. Expression vectors were amplified and purified by Plasmid Midi Kit (QIAGEN, Germany).

    Article Title: Time-Restricted PiggyBac DNA Transposition by Transposase Protein Delivery Using Lentivirus-Derived Nanoparticles
    Article Snippet: .. Purified DNA was ligated overnight with T4 DNA ligase (New England Biolabs) in a total volume of 500 μL. .. 2 μL ligated DNA was used as template for the first PCR using DreamTaq DNA polymerase (Thermo Fisher Scientific) following manufacturer’s instructions (57°C annealing temperature; 2 min extension time; 50 μL total volume) with a piggyBac-specific sense primer and a PGK-specific antisense primer.

    Polymerase Chain Reaction:

    Article Title: SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome
    Article Snippet: PCR products were then restricted using EcoRI and BamHI enzymes and ligated into pEGFP-N2 expression vector. .. Ligations were done using T4 DNA ligase (New England Biolabs).

    Article Title: Silencing of Rieske Iron-Sulfur Protein Impacts Upon the Development and Reproduction of Spodoptera exigua by Regulating ATP Synthesis
    Article Snippet: PCR products from RISPs PCRs, and the pET32a expression vectors, were separately digested using Sac I and Xho I (New England BioLabs). .. Then, 50 μL digestion reactions were set up consisting of 2 μg of DNA, 5 μL of 10 × NEB buffer 4, 1 μL of 100 × BSA and 1 μL of 20 U/μL enzyme; these were incubated at 37°C for 4 h. Digested RISPs and pET32a expression vectors were then purified using a Universal DNA Purification Kit (TIANGEN) in accordance with the manufacturer's recommendations and then ligated together using T4 DNA Ligase (New England BioLabs).

    Article Title: CRISPR-FRT targets shared sites in a knock-out collection for off-the-shelf genome editing
    Article Snippet: .. However, in the latter case the resulting PCR product was cut with SpeI (NEB), the ends self-ligated with T4 DNA ligase (New England Biolabs), phenol:chloroform extracted, ethanol precipitated and transformed into DH5α cells. ..

    Article Title: FdC1 and Leaf-Type Ferredoxins Channel Electrons From Photosystem I to Different Downstream Electron Acceptors
    Article Snippet: .. The restricted PCR products were ligated into corresponding vectors by the T4 DNA ligase (NEB, United States). .. Subcellular Localization of FdC1 in Intact Tobacco Leaves The full-length coding sequence of FdC1 was amplified and cloned into the XbaI site of the pBA002 vector which expresses an enhanced yellow florescence protein (eYFP) under the control of a CaMV35S promoter ( ).

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: KK8235) PEG/NaCl SPRI solution, supplied with HTP Library Preparation Kit PCR-free (96rxn) (Kapa Biosystems) 2× Kapa HiFi HotStart Ready Mix (Kapa Biosystems, cat.no. .. M0202L) 10× T4 DNA ligase Buffer (New England BioLabs), supplied with T4 DNA Ligase USER Enzyme (New England BioLabs, cat.no.

    Article Title: Time-Restricted PiggyBac DNA Transposition by Transposase Protein Delivery Using Lentivirus-Derived Nanoparticles
    Article Snippet: Purified DNA was ligated overnight with T4 DNA ligase (New England Biolabs) in a total volume of 500 μL. .. 2 μL ligated DNA was used as template for the first PCR using DreamTaq DNA polymerase (Thermo Fisher Scientific) following manufacturer’s instructions (57°C annealing temperature; 2 min extension time; 50 μL total volume) with a piggyBac-specific sense primer and a PGK-specific antisense primer.

    Article Title: Biosynthesis of thiocarboxylic acid-containing natural products
    Article Snippet: PCR primers were obtained from Sigma-Aldrich. .. Q5 high-fidelity DNA polymerase, restriction endonucleases, and T4 DNA ligase were purchased from NEB and used by following the protocols provided by the manufacturers.

    CRISPR:

    Article Title: Crystal structure of intraflagellar transport protein 80 reveals a homo-dimer required for ciliogenesis
    Article Snippet: Paragraph title: CRISPR/Cas9 generation of mutations in Ift80 ... Ligations were carried out in 10 µl reactions in 1X T4 DNA ligase buffer (NEB), using 50 ng linear pX330, 1 µl annealed oligos (0.5 µM) and 1 µl T4 DNA ligase (NEB), and were incubated at 4°C for 16 hr.

    Nested PCR:

    Article Title: Time-Restricted PiggyBac DNA Transposition by Transposase Protein Delivery Using Lentivirus-Derived Nanoparticles
    Article Snippet: Purified DNA was ligated overnight with T4 DNA ligase (New England Biolabs) in a total volume of 500 μL. .. 2 μL of the first round of PCR was used as a template for a nested PCR reaction employing identical settings as the first-round PCR.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: .. M0202L) 10× T4 DNA ligase Buffer (New England BioLabs), supplied with T4 DNA ligase LB agar (Miller powder) (Fisher Scientific, cat.no. .. DF0445174) Luria Broth base (Miller’s LB Broth Base) (Thermo Fisher Scientific, cat.no.

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: .. M0202L) 10× T4 DNA ligase Buffer (New England BioLabs), supplied with T4 DNA Ligase USER Enzyme (New England BioLabs, cat.no. .. M5505L) Exonuclease I ( E. coli ) (New England BioLabs, cat.no.

    Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals
    Article Snippet: The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; M0202) ligation protocol. .. The pWPT-ΔGFP-X plasmids encoding the native form of the X proteins were obtained after digestion of the pWPT-GFP-X vectors using MluI and NotI and amplification of each X using Mlu1-pWPT-X-F primer (5′-GCT TAC GCG TTC TGC AGT CGA CGA ATT CAC CAT G-3′) and pWPT-R primer (5′-GTC AGC AAA CAC AGT GCA CAC CA-3′).

    Plasmid Preparation:

    Article Title: SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome
    Article Snippet: PCR products were then restricted using EcoRI and BamHI enzymes and ligated into pEGFP-N2 expression vector. .. Ligations were done using T4 DNA ligase (New England Biolabs).

    Article Title: Silencing of Rieske Iron-Sulfur Protein Impacts Upon the Development and Reproduction of Spodoptera exigua by Regulating ATP Synthesis
    Article Snippet: Then, 50 μL digestion reactions were set up consisting of 2 μg of DNA, 5 μL of 10 × NEB buffer 4, 1 μL of 100 × BSA and 1 μL of 20 U/μL enzyme; these were incubated at 37°C for 4 h. Digested RISPs and pET32a expression vectors were then purified using a Universal DNA Purification Kit (TIANGEN) in accordance with the manufacturer's recommendations and then ligated together using T4 DNA Ligase (New England BioLabs). .. The 10 μL ligation reaction consisted of 5 μL of purified RISPs, 3 μL of purified vector, 1 μL of 10 × T4 DNA ligase buffer, and 1 μL of T4 DNA ligase.

    Article Title: CRISPR-FRT targets shared sites in a knock-out collection for off-the-shelf genome editing
    Article Snippet: Paragraph title: Plasmid and strain construction ... However, in the latter case the resulting PCR product was cut with SpeI (NEB), the ends self-ligated with T4 DNA ligase (New England Biolabs), phenol:chloroform extracted, ethanol precipitated and transformed into DH5α cells.

    Article Title: Crystal structure of intraflagellar transport protein 80 reveals a homo-dimer required for ciliogenesis
    Article Snippet: 100 ng circular plasmid pX330 (Addgene ID 42230) was digested with 4.0 µl BbsI (5000 U/ml) restriction enzyme (NEB, Ipswich, MA) in a 50 µl reaction with 1X NEBuffer 2.1 (NEB), for 1 hr at 37°C. .. Ligations were carried out in 10 µl reactions in 1X T4 DNA ligase buffer (NEB), using 50 ng linear pX330, 1 µl annealed oligos (0.5 µM) and 1 µl T4 DNA ligase (NEB), and were incubated at 4°C for 16 hr.

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: M0202L) 10× T4 DNA ligase Buffer (New England BioLabs), supplied with T4 DNA Ligase USER Enzyme (New England BioLabs, cat.no. .. M0262L) Plasmid-Safe ATP-dependent DNase (Epicentre, cat.no.

    Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals
    Article Snippet: The X coding regions (synthesized by Genewiz) from hepadnaviruses infecting the New World wooly monkey ( Lagothrix ) (WMHBx) and three distant bat species, including the roundleaf bat ( Hipposideros cf. ruber ), the horseshoe bat ( Rhinolophus Alcyone ), and the tent-making bat ( Uroderma bilobatum ) (RBHBx, HBHBx, TBHBx, respectively) , were expressed from the same vector. .. The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; M0202) ligation protocol.

    Article Title: A Single Codon Optimization Enhances Recombinant Human TNF-α Vaccine Expression in Escherichia coli
    Article Snippet: Reagents Restriction enzymes, T4 DNA ligase, and Phusion High-Fidelity DNA Polymerase were purchased from New England Biolabs (Ipswich, MA). .. The vector pET-22b and E. coli strain BL21 (DE3) were purchased from Novagen (San Diego, CA).

    Article Title: Biosynthesis of thiocarboxylic acid-containing natural products
    Article Snippet: Q5 high-fidelity DNA polymerase, restriction endonucleases, and T4 DNA ligase were purchased from NEB and used by following the protocols provided by the manufacturers. .. DNA gel extraction and plasmid preparation kits were purchased from Omega Bio-Tek.

    Article Title: Translation efficiency of heterologous proteins is significantly affected by the genetic context of RBS sequences in engineered cyanobacterium Synechocystis sp. PCC 6803
    Article Snippet: Enzymes and reagents The restriction enzymes, T4 DNA ligase and DNA polymerase used in this work were purchased from New England BioLabs (USA) or from ThermoFischer-Scientific (USA). .. Commercial Qiagen (Germany) kits were used for plasmid isolation (QIAprep Spin miniprep kit) and gel extraction (QIAquick, gel extraction kit).

    Negative Control:

    Article Title: Silencing of Rieske Iron-Sulfur Protein Impacts Upon the Development and Reproduction of Spodoptera exigua by Regulating ATP Synthesis
    Article Snippet: Then, 50 μL digestion reactions were set up consisting of 2 μg of DNA, 5 μL of 10 × NEB buffer 4, 1 μL of 100 × BSA and 1 μL of 20 U/μL enzyme; these were incubated at 37°C for 4 h. Digested RISPs and pET32a expression vectors were then purified using a Universal DNA Purification Kit (TIANGEN) in accordance with the manufacturer's recommendations and then ligated together using T4 DNA Ligase (New England BioLabs). .. Then, the reaction tubes were incubated at 16°C for 12 h. Recombinant plasmids (pET32a-RISPs) were then transformed into Escherichia coli Transetta (DE3), using a non-carrier pET32a vector as a negative control.

    Positron Emission Tomography:

    Article Title: A Single Codon Optimization Enhances Recombinant Human TNF-α Vaccine Expression in Escherichia coli
    Article Snippet: Reagents Restriction enzymes, T4 DNA ligase, and Phusion High-Fidelity DNA Polymerase were purchased from New England Biolabs (Ipswich, MA). .. The vector pET-22b and E. coli strain BL21 (DE3) were purchased from Novagen (San Diego, CA).

    In Situ:

    Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies
    Article Snippet: Paragraph title: In situ Hi-C of knockdown and control cells ... Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U T4 DNA ligase NEB M0202S, final sample volume 1.2 ml) and samples were incubated for 4 h at room temperature under rotation.

    Next-Generation Sequencing:

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: Paragraph title: CIRCLE-seq library preparation and NGS ... M0202L) 10× T4 DNA ligase Buffer (New England BioLabs), supplied with T4 DNA Ligase USER Enzyme (New England BioLabs, cat.no.

    Concentration Assay:

    Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies
    Article Snippet: After 10 min incubation at room temperature, SDS was quenched adding 1% Triton X-100 (final concentration) and 1X of NEBuffer 3.1 (NEB, B7203S). .. Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U T4 DNA ligase NEB M0202S, final sample volume 1.2 ml) and samples were incubated for 4 h at room temperature under rotation.

    Article Title: Crystal structure of intraflagellar transport protein 80 reveals a homo-dimer required for ciliogenesis
    Article Snippet: At a concentration of 100 µM, oligos were annealed in a thermocycler: 37°C, 30 min; 95°C, 5 min; ramp down to 25°C at −5 °C/min. .. Ligations were carried out in 10 µl reactions in 1X T4 DNA ligase buffer (NEB), using 50 ng linear pX330, 1 µl annealed oligos (0.5 µM) and 1 µl T4 DNA ligase (NEB), and were incubated at 4°C for 16 hr.

    DNA Purification:

    Article Title: Silencing of Rieske Iron-Sulfur Protein Impacts Upon the Development and Reproduction of Spodoptera exigua by Regulating ATP Synthesis
    Article Snippet: .. Then, 50 μL digestion reactions were set up consisting of 2 μg of DNA, 5 μL of 10 × NEB buffer 4, 1 μL of 100 × BSA and 1 μL of 20 U/μL enzyme; these were incubated at 37°C for 4 h. Digested RISPs and pET32a expression vectors were then purified using a Universal DNA Purification Kit (TIANGEN) in accordance with the manufacturer's recommendations and then ligated together using T4 DNA Ligase (New England BioLabs). .. The 10 μL ligation reaction consisted of 5 μL of purified RISPs, 3 μL of purified vector, 1 μL of 10 × T4 DNA ligase buffer, and 1 μL of T4 DNA ligase.

    Gel Extraction:

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: R3535L) 10× CutSmart Buffer (New England BioLabs) supplied with Bsa I-HF QIAquick Gel extraction kit (Qiagen, cat.no. .. M0202L) 10× T4 DNA ligase Buffer (New England BioLabs), supplied with T4 DNA ligase LB agar (Miller powder) (Fisher Scientific, cat.no.

    Article Title: Time-Restricted PiggyBac DNA Transposition by Transposase Protein Delivery Using Lentivirus-Derived Nanoparticles
    Article Snippet: Gel Extraction Kit (Omega Bio-tek, Norcross, GA). .. Purified DNA was ligated overnight with T4 DNA ligase (New England Biolabs) in a total volume of 500 μL.

    Article Title: Biosynthesis of thiocarboxylic acid-containing natural products
    Article Snippet: Q5 high-fidelity DNA polymerase, restriction endonucleases, and T4 DNA ligase were purchased from NEB and used by following the protocols provided by the manufacturers. .. DNA gel extraction and plasmid preparation kits were purchased from Omega Bio-Tek.

    Article Title: Translation efficiency of heterologous proteins is significantly affected by the genetic context of RBS sequences in engineered cyanobacterium Synechocystis sp. PCC 6803
    Article Snippet: Enzymes and reagents The restriction enzymes, T4 DNA ligase and DNA polymerase used in this work were purchased from New England BioLabs (USA) or from ThermoFischer-Scientific (USA). .. Commercial Qiagen (Germany) kits were used for plasmid isolation (QIAprep Spin miniprep kit) and gel extraction (QIAquick, gel extraction kit).

    Homologous Recombination:

    Article Title: Biosynthesis of thiocarboxylic acid-containing natural products
    Article Snippet: Q5 high-fidelity DNA polymerase, restriction endonucleases, and T4 DNA ligase were purchased from NEB and used by following the protocols provided by the manufacturers. .. The REDIRECT Technology kit for PCR-targeting homologous recombination was provided by The John Innes Center (Norwich, UK) . pOJ260 was used as a shuttle vector for gene homologous recombination .

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    New England Biolabs m0202 ligation protocol
    Effect of Inhibiting dsDNA on Enzyme Self-Adenylylation Rate. The determined rates for self-adenylylation of an uninhibited reaction, 2.5 μM <t>T4</t> DNA ligase (red) and 2.5 μM T4 DNA ligase and inhibited reactions 2.5 μM DNA (blue) and 10 μM DNA (green). The reactions were fit to a single exponential equation ( Eq 6 ) to determine the reaction rate. The uninhibited reaction was determined to have a single turnover rate of 20 s -1 ± 2 s -1 . While the 2.5 μM inhibited reaction had a single turnover rate of 2.8 s -1 ± 0.5 s -1 and the 10 μM inhibited reaction had a single turnover rate of 1.0 s -1 ± 1 s -1 . All reactions were performed a minimum of three times at 16°C. Error reported is the standard error for the replicates.
    M0202 Ligation Protocol, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of Inhibiting dsDNA on Enzyme Self-Adenylylation Rate. The determined rates for self-adenylylation of an uninhibited reaction, 2.5 μM T4 DNA ligase (red) and 2.5 μM T4 DNA ligase and inhibited reactions 2.5 μM DNA (blue) and 10 μM DNA (green). The reactions were fit to a single exponential equation ( Eq 6 ) to determine the reaction rate. The uninhibited reaction was determined to have a single turnover rate of 20 s -1 ± 2 s -1 . While the 2.5 μM inhibited reaction had a single turnover rate of 2.8 s -1 ± 0.5 s -1 and the 10 μM inhibited reaction had a single turnover rate of 1.0 s -1 ± 1 s -1 . All reactions were performed a minimum of three times at 16°C. Error reported is the standard error for the replicates.

    Journal: PLoS ONE

    Article Title: The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    doi: 10.1371/journal.pone.0150802

    Figure Lengend Snippet: Effect of Inhibiting dsDNA on Enzyme Self-Adenylylation Rate. The determined rates for self-adenylylation of an uninhibited reaction, 2.5 μM T4 DNA ligase (red) and 2.5 μM T4 DNA ligase and inhibited reactions 2.5 μM DNA (blue) and 10 μM DNA (green). The reactions were fit to a single exponential equation ( Eq 6 ) to determine the reaction rate. The uninhibited reaction was determined to have a single turnover rate of 20 s -1 ± 2 s -1 . While the 2.5 μM inhibited reaction had a single turnover rate of 2.8 s -1 ± 0.5 s -1 and the 10 μM inhibited reaction had a single turnover rate of 1.0 s -1 ± 1 s -1 . All reactions were performed a minimum of three times at 16°C. Error reported is the standard error for the replicates.

    Article Snippet: DsDNA inhibition likely also has an effect in commonly used molecular biology protocols, for example, the maximal recommended DNA concentration utilized in the ligation step for Next Generation Sequencing library preparation is ~20 ng/μL (NEB Ultra II), while the recommended DNA concentration in a standard sticky-end ligation is 4.38 ng/μL (NEB T4 DNA ligase ligation protocol).

    Techniques:

    T4 DNA Ligase Reaction Model. Modified reaction pathway to include the newly observed reactions in the previously described DNA ligation pathway that are inhibited by the presence of non-nicked dsDNA. A . Non-nicked dsDNA can bind to the deadenylylated form of the enzyme inhibition formation of the adenylylated form of the enzyme. B . Non-nicked dsDNA binds to the Lig-AMP form, preventing complexation with its preferred ds-nDNA substrate.

    Journal: PLoS ONE

    Article Title: The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    doi: 10.1371/journal.pone.0150802

    Figure Lengend Snippet: T4 DNA Ligase Reaction Model. Modified reaction pathway to include the newly observed reactions in the previously described DNA ligation pathway that are inhibited by the presence of non-nicked dsDNA. A . Non-nicked dsDNA can bind to the deadenylylated form of the enzyme inhibition formation of the adenylylated form of the enzyme. B . Non-nicked dsDNA binds to the Lig-AMP form, preventing complexation with its preferred ds-nDNA substrate.

    Article Snippet: DsDNA inhibition likely also has an effect in commonly used molecular biology protocols, for example, the maximal recommended DNA concentration utilized in the ligation step for Next Generation Sequencing library preparation is ~20 ng/μL (NEB Ultra II), while the recommended DNA concentration in a standard sticky-end ligation is 4.38 ng/μL (NEB T4 DNA ligase ligation protocol).

    Techniques: Modification, DNA Ligation, Enzyme Inhibition Assay

    k cat /K m Curve for T4 DNA Ligase. The data was obtained through titration of increasing concentrations of a 75mer-ds-nDNA substrate, reacted at 16°C to determine initial reaction rates. T4 DNA ligase concentrations used were 25 pM– 100 pM. The initial rates were plotted against their respective substrate concentrations and fit by: A . a classical uncompetitive substrate inhibition model ( Eq 2 ), where k cat and K m Values of 0.44 s -1 ± 0.3 s -1 and 4 nM ± 1 nM respectively, were determined. The K i value for substrate inhibition was calculated to be 590 nM ± 170 nM. B . A competitive substrate inhibition for a Bi-Bi Ping-Pong mechanism ( Eq 3 ) k cat and K m values of 0.48 s -1 ± 0.3 s -1 and 4 nM ± 1 nM respectively, were determined. The K i value for substrate inhibition was calculated to be 54 nM ± 15 nM. All data points are the average of at least three independent experiments, and the error reported is the standard deviation for the replicates.

    Journal: PLoS ONE

    Article Title: The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    doi: 10.1371/journal.pone.0150802

    Figure Lengend Snippet: k cat /K m Curve for T4 DNA Ligase. The data was obtained through titration of increasing concentrations of a 75mer-ds-nDNA substrate, reacted at 16°C to determine initial reaction rates. T4 DNA ligase concentrations used were 25 pM– 100 pM. The initial rates were plotted against their respective substrate concentrations and fit by: A . a classical uncompetitive substrate inhibition model ( Eq 2 ), where k cat and K m Values of 0.44 s -1 ± 0.3 s -1 and 4 nM ± 1 nM respectively, were determined. The K i value for substrate inhibition was calculated to be 590 nM ± 170 nM. B . A competitive substrate inhibition for a Bi-Bi Ping-Pong mechanism ( Eq 3 ) k cat and K m values of 0.48 s -1 ± 0.3 s -1 and 4 nM ± 1 nM respectively, were determined. The K i value for substrate inhibition was calculated to be 54 nM ± 15 nM. All data points are the average of at least three independent experiments, and the error reported is the standard deviation for the replicates.

    Article Snippet: DsDNA inhibition likely also has an effect in commonly used molecular biology protocols, for example, the maximal recommended DNA concentration utilized in the ligation step for Next Generation Sequencing library preparation is ~20 ng/μL (NEB Ultra II), while the recommended DNA concentration in a standard sticky-end ligation is 4.38 ng/μL (NEB T4 DNA ligase ligation protocol).

    Techniques: Titration, Inhibition, Standard Deviation

    Various Inhibitors Effects on Rate of Nick Sealing. Various concentrations of dsDNA substrates were utilized as potential inhibitors of the T4 DNA ligase steady state ligation reaction on 20 nM of the 75mer-ds-nDNA substrate. All reactions were performed in the presence of 25 pM of T4 DNA ligase, a minimum of three times at 16°C. Error reported is the standard deviation for the replicates.

    Journal: PLoS ONE

    Article Title: The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    doi: 10.1371/journal.pone.0150802

    Figure Lengend Snippet: Various Inhibitors Effects on Rate of Nick Sealing. Various concentrations of dsDNA substrates were utilized as potential inhibitors of the T4 DNA ligase steady state ligation reaction on 20 nM of the 75mer-ds-nDNA substrate. All reactions were performed in the presence of 25 pM of T4 DNA ligase, a minimum of three times at 16°C. Error reported is the standard deviation for the replicates.

    Article Snippet: DsDNA inhibition likely also has an effect in commonly used molecular biology protocols, for example, the maximal recommended DNA concentration utilized in the ligation step for Next Generation Sequencing library preparation is ~20 ng/μL (NEB Ultra II), while the recommended DNA concentration in a standard sticky-end ligation is 4.38 ng/μL (NEB T4 DNA ligase ligation protocol).

    Techniques: Ligation, Standard Deviation

    Competition for ds-nDNA-Binding by dsDNA. Lane one contains 4 nM of the 75mer-ds-nDNA substrate alone, lanes 2–6 show shifting of the 4 nM substrate into a completely bound state as the concentration of T4 DNA ligase is increased from 100 nM– 1000 nM. Lanes 7–11 are of a titration of increasing concentrations of the unlabeled I-75-dsDNA oligo into a reaction containing 4 nM labeled nicked substrate and 1000 nM T4 DNA ligase. EMSA reactions were all performed and electrophoresed at room temperature (22°C).

    Journal: PLoS ONE

    Article Title: The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    doi: 10.1371/journal.pone.0150802

    Figure Lengend Snippet: Competition for ds-nDNA-Binding by dsDNA. Lane one contains 4 nM of the 75mer-ds-nDNA substrate alone, lanes 2–6 show shifting of the 4 nM substrate into a completely bound state as the concentration of T4 DNA ligase is increased from 100 nM– 1000 nM. Lanes 7–11 are of a titration of increasing concentrations of the unlabeled I-75-dsDNA oligo into a reaction containing 4 nM labeled nicked substrate and 1000 nM T4 DNA ligase. EMSA reactions were all performed and electrophoresed at room temperature (22°C).

    Article Snippet: DsDNA inhibition likely also has an effect in commonly used molecular biology protocols, for example, the maximal recommended DNA concentration utilized in the ligation step for Next Generation Sequencing library preparation is ~20 ng/μL (NEB Ultra II), while the recommended DNA concentration in a standard sticky-end ligation is 4.38 ng/μL (NEB T4 DNA ligase ligation protocol).

    Techniques: Binding Assay, Concentration Assay, Titration, Labeling