t4 dna ligase ligated plasmids  (Thermo Fisher)


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    Name:
    T4 DNA Ligase
    Description:
    Thermo Scientific T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex DNA or RNA The enzyme repairs single strand nicks in duplex DNA RNA or DNA RNA hybrids It also joins DNA fragments with either cohesive or blunt termini but has no activity on single stranded nucleic acids T4 DNA Ligase requires ATP as a cofactor Highlights• Active in Themo Scientific restriction enzyme PCR and RT buffers when supplemented with ATP • Fast sticky end ligation is completed in 10 minutes at room temperature• Supplied with PEG solution for efficient blunt end ligationApplications• Cloning of restriction enzyme generated DNA fragments• Cloning of PCR products• Joining of double stranded oligonucleotide linkers or adaptors to DNA• Site directed mutagenesis• Amplified fragment length polymorphism AFLP • Ligase mediated RNA detection see Reference 3 • Nick repair in duplex DNA RNA or DNA RNA hybrids• Self circularization of linear DNA Includes• T4 DNA Ligase• 10X T4 DNA Ligase Buffer• 50 PEG SolutionNotes• Binding of T4 DNA Ligase to DNA may result in a band shift in agarose gels To avoid this incubate samples with 6X DNA Loading Dye SDS Solution at 70°C for 5 min or 65°C for 10 minutes and chill on ice prior to electrophoresis • The volume of the ligation reaction mixture should not exceed 10 of the competent cell volume in the transformation process • Prior to electro transformation remove T4 DNA Ligase from the ligation mixture using spin columns or chloroform extraction The extracted DNA can be further precipitated with ethanol
    Catalog Number:
    el0013
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher t4 dna ligase ligated plasmids
    Thermo Scientific T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex DNA or RNA The enzyme repairs single strand nicks in duplex DNA RNA or DNA RNA hybrids It also joins DNA fragments with either cohesive or blunt termini but has no activity on single stranded nucleic acids T4 DNA Ligase requires ATP as a cofactor Highlights• Active in Themo Scientific restriction enzyme PCR and RT buffers when supplemented with ATP • Fast sticky end ligation is completed in 10 minutes at room temperature• Supplied with PEG solution for efficient blunt end ligationApplications• Cloning of restriction enzyme generated DNA fragments• Cloning of PCR products• Joining of double stranded oligonucleotide linkers or adaptors to DNA• Site directed mutagenesis• Amplified fragment length polymorphism AFLP • Ligase mediated RNA detection see Reference 3 • Nick repair in duplex DNA RNA or DNA RNA hybrids• Self circularization of linear DNA Includes• T4 DNA Ligase• 10X T4 DNA Ligase Buffer• 50 PEG SolutionNotes• Binding of T4 DNA Ligase to DNA may result in a band shift in agarose gels To avoid this incubate samples with 6X DNA Loading Dye SDS Solution at 70°C for 5 min or 65°C for 10 minutes and chill on ice prior to electrophoresis • The volume of the ligation reaction mixture should not exceed 10 of the competent cell volume in the transformation process • Prior to electro transformation remove T4 DNA Ligase from the ligation mixture using spin columns or chloroform extraction The extracted DNA can be further precipitated with ethanol
    https://www.bioz.com/result/t4 dna ligase ligated plasmids/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase ligated plasmids - by Bioz Stars, 2020-08
    99/100 stars

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    Related Articles

    Nucleic Acid Electrophoresis:

    Article Title: Reverse transcriptase and endonuclease activities encoded by Penelope-like retroelements
    Article Snippet: .. After addition of 0.5 Weiss unit of T4 DNA ligase (Fermentas, Hanover, MD), reactions were incubated at 16°C and analyzed by denaturing gel electrophoresis as above. ..

    Synthesized:

    Article Title: Practical Synthesis of Cap‐4 RNA
    Article Snippet: .. The 38‐nt T. cruzi cap‐4 RNA was prepared by splinted enzymatic ligation of an 11‐nt cap‐4 RNA and a chemically synthesized 5′‐phosphorylated 27‐nt RNA by using T4 DNA ligase (Thermo Fisher) in analogy to ref. . ..

    Ligation:

    Article Title: Identification of recognition residues for ligation-based detection and quantitation of pseudouridine and N6-methyladenosine
    Article Snippet: .. The optimized ligation reactions were carried out with 0.15 µM 30-mer RNA with or without Ψ or m6 A modifications, 0.5 µM floater and 0.38 µM of 5′-32 P-labeled anchor in 66 mM Tris–HCl, pH 7.6, 0.5 mM ZnCl2 , 10 mM DTT, 66 µM ATP, 15% DMSO and 0.25 U/µl T4 DNA ligase (USB Inc.). .. All components were mixed and incubated at 16°C for 16 h, and the ligation products separated on denaturing polyacrylamide gels containing 7 M urea.

    Article Title: Practical Synthesis of Cap‐4 RNA
    Article Snippet: .. The 38‐nt T. cruzi cap‐4 RNA was prepared by splinted enzymatic ligation of an 11‐nt cap‐4 RNA and a chemically synthesized 5′‐phosphorylated 27‐nt RNA by using T4 DNA ligase (Thermo Fisher) in analogy to ref. . ..

    Article Title: Profiling non-lysyl tRNAs in HIV-1
    Article Snippet: .. The ligation reaction was carried out overnight (∼16 h) at 16°C with 0.13 μg/μL total RNA in 1X T4 DNA ligase buffer, 0.5 U/μL T4 DNA ligase (USB Corporation, 70042X), 15% DMSO, and 4.5 μM labeling oligonucleotide. .. Hybridization was performed at 60°C overnight (∼16 h) with 1–2 μg each of Cy3- or Cy5-labeled total RNA as previously described ( ).

    Labeling:

    Article Title: Profiling non-lysyl tRNAs in HIV-1
    Article Snippet: .. The ligation reaction was carried out overnight (∼16 h) at 16°C with 0.13 μg/μL total RNA in 1X T4 DNA ligase buffer, 0.5 U/μL T4 DNA ligase (USB Corporation, 70042X), 15% DMSO, and 4.5 μM labeling oligonucleotide. .. Hybridization was performed at 60°C overnight (∼16 h) with 1–2 μg each of Cy3- or Cy5-labeled total RNA as previously described ( ).

    Purification:

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    Article Snippet: .. First, the purified product of PCR for the FSH1 gene was ligated into pUC-PUT following DNA digestion with Xho I and Hin dIII, and ligated by T4 DNA ligase (Invitrogen; Thermo Fisher Scientific, Inc.). ..

    Incubation:

    Article Title: Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends
    Article Snippet: .. Reactions were started by the addition of 0.5 U of T4 DNA ligase (Invitrogen, Carlsbad, CA) and incubated at 37°C. ..

    Article Title: Reverse transcriptase and endonuclease activities encoded by Penelope-like retroelements
    Article Snippet: .. After addition of 0.5 Weiss unit of T4 DNA ligase (Fermentas, Hanover, MD), reactions were incubated at 16°C and analyzed by denaturing gel electrophoresis as above. ..

    Polymerase Chain Reaction:

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    Article Snippet: .. First, the purified product of PCR for the FSH1 gene was ligated into pUC-PUT following DNA digestion with Xho I and Hin dIII, and ligated by T4 DNA ligase (Invitrogen; Thermo Fisher Scientific, Inc.). ..

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    Thermo Fisher t4 dna ligase
    In vitro seamless assembly of the whole actinorhodin biosynthetic cluster from multiple restriction fragments. ( A ) The schematic diagram of the PCR amplicons. The 29-kb actinorhodin biosynthetic cluster was divided into four fragments, which were then PCR amplified with primers specified ( Supplementary Table S1 ). ( B ) Errorless demethylated fragments I (7420 bp), II (8171 bp), III (6410 bp) and IV (6686 bp) were released from pBluescript II KS by XbaI digestion, which were then ligated with a designed adaptor klf.ML2 ( Figure 1 B and Supplementary Table S1 ) and digested with MspJI as described in the ‘Materials and Methods’ section. The MspJI-treated fragments were used for ligation with <t>T4</t> DNA ligase at 16°C for 2 h. ( C ) Fragments I–II and III–IV were ligated with pHI with (‘L+’) or without (‘L−’) the addition of T4 DNA ligase. The synthesized DNA could be viewed in the ‘L+’ lane together with the disappearance of the substrate fragments. The ligation reaction was performed at temperature 22°C for 8 h. ( D ) The BamHI restriction map of the synthesized plasmid pHIW. The theoretical restriction fragments of pHIW contains fragments of 11 848, 7001, 5804, 3923, 2172, 1731, 1191, 1029, 928 and 743 bp in size. The 743-bp fragment was run out of the gel and was not shown. ( E ) Heterologous expression of the assembled actinorhodin biosynthetic cluster in Streptomyces strain 4F (4F/pHIW), using 4F integrated plasmid pHI (4F/pHI) as a negative control. Strains were cultured on R2YE medium at 30°C for 2 days.
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 550 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/Thermo Fisher
    Average 99 stars, based on 550 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2020-08
    99/100 stars
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    99
    Thermo Fisher t4 ligase
    Summary of the Golden GATEway cloning kit. A) Outline of the entire cloning procedure. Eight different entry vectors (pGG-EVx) contain different inserts (colored bars). These inserts are assembled in a predefined order using a Golden Gate reaction into Gateway TM  entry vectors at any position (Threeway Gateway TM  cloning). These can then be assembled to establish a final expression vector in an LR reaction. Compatible overhangs are indicated on each Golden Gate entry vector. B) The principle of Golden Gate cloning is illustrated in the top scheme; the principle of Gateway cloning is illustrated in the bottom scheme. Golden Gate cloning utilizes type II restriction endonucleases to generate compatible overhangs that can be ligated with T4 ligase. The ligation of the two compatible inserts from the entry vectors one and two is illustrated. Gateway cloning relies on recombination of specific att sites using a commercially available enzyme mix (LR Clonase II, Life Technologies).
    T4 Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 527 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 ligase/product/Thermo Fisher
    Average 99 stars, based on 527 article reviews
    Price from $9.99 to $1999.99
    t4 ligase - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

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    In vitro seamless assembly of the whole actinorhodin biosynthetic cluster from multiple restriction fragments. ( A ) The schematic diagram of the PCR amplicons. The 29-kb actinorhodin biosynthetic cluster was divided into four fragments, which were then PCR amplified with primers specified ( Supplementary Table S1 ). ( B ) Errorless demethylated fragments I (7420 bp), II (8171 bp), III (6410 bp) and IV (6686 bp) were released from pBluescript II KS by XbaI digestion, which were then ligated with a designed adaptor klf.ML2 ( Figure 1 B and Supplementary Table S1 ) and digested with MspJI as described in the ‘Materials and Methods’ section. The MspJI-treated fragments were used for ligation with T4 DNA ligase at 16°C for 2 h. ( C ) Fragments I–II and III–IV were ligated with pHI with (‘L+’) or without (‘L−’) the addition of T4 DNA ligase. The synthesized DNA could be viewed in the ‘L+’ lane together with the disappearance of the substrate fragments. The ligation reaction was performed at temperature 22°C for 8 h. ( D ) The BamHI restriction map of the synthesized plasmid pHIW. The theoretical restriction fragments of pHIW contains fragments of 11 848, 7001, 5804, 3923, 2172, 1731, 1191, 1029, 928 and 743 bp in size. The 743-bp fragment was run out of the gel and was not shown. ( E ) Heterologous expression of the assembled actinorhodin biosynthetic cluster in Streptomyces strain 4F (4F/pHIW), using 4F integrated plasmid pHI (4F/pHI) as a negative control. Strains were cultured on R2YE medium at 30°C for 2 days.

    Journal: Nucleic Acids Research

    Article Title: The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly

    doi: 10.1093/nar/gkt122

    Figure Lengend Snippet: In vitro seamless assembly of the whole actinorhodin biosynthetic cluster from multiple restriction fragments. ( A ) The schematic diagram of the PCR amplicons. The 29-kb actinorhodin biosynthetic cluster was divided into four fragments, which were then PCR amplified with primers specified ( Supplementary Table S1 ). ( B ) Errorless demethylated fragments I (7420 bp), II (8171 bp), III (6410 bp) and IV (6686 bp) were released from pBluescript II KS by XbaI digestion, which were then ligated with a designed adaptor klf.ML2 ( Figure 1 B and Supplementary Table S1 ) and digested with MspJI as described in the ‘Materials and Methods’ section. The MspJI-treated fragments were used for ligation with T4 DNA ligase at 16°C for 2 h. ( C ) Fragments I–II and III–IV were ligated with pHI with (‘L+’) or without (‘L−’) the addition of T4 DNA ligase. The synthesized DNA could be viewed in the ‘L+’ lane together with the disappearance of the substrate fragments. The ligation reaction was performed at temperature 22°C for 8 h. ( D ) The BamHI restriction map of the synthesized plasmid pHIW. The theoretical restriction fragments of pHIW contains fragments of 11 848, 7001, 5804, 3923, 2172, 1731, 1191, 1029, 928 and 743 bp in size. The 743-bp fragment was run out of the gel and was not shown. ( E ) Heterologous expression of the assembled actinorhodin biosynthetic cluster in Streptomyces strain 4F (4F/pHIW), using 4F integrated plasmid pHI (4F/pHI) as a negative control. Strains were cultured on R2YE medium at 30°C for 2 days.

    Article Snippet: GeneRuler™ 1-kb DNA ladder (250–10 000 bp), thermosensitive alkaline phosphatase (FastAP™) and T4 DNA ligase were purchased from Thermo Fermentas.

    Techniques: In Vitro, Polymerase Chain Reaction, Amplification, Ligation, Synthesized, Plasmid Preparation, Expressing, Negative Control, Cell Culture

    Ligation independent cloning strategy. Flanking regions (350 bp and 500 bp) of the 10 AA containing active site were amplified from genomic DNA with tailed primers, introducing restriction sites that enabled restriction and ligation into the pUC 18. After restriction with PstI, a mixture of linear plasmid DNA , amplified synthetic DNA fragment and T4 DNA polymerase was prepared, as proposed by Thieme et al . ( 2011 ). The mixture was incubated at 25°C for 5 min, and subsequently used for transformation of E. coli Top10 cells (Invitrogen ™ ). Correct constructs were obtained with very high efficiencies (80–95%).

    Journal: MicrobiologyOpen

    Article Title: Systematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach

    doi: 10.1002/mbo3.326

    Figure Lengend Snippet: Ligation independent cloning strategy. Flanking regions (350 bp and 500 bp) of the 10 AA containing active site were amplified from genomic DNA with tailed primers, introducing restriction sites that enabled restriction and ligation into the pUC 18. After restriction with PstI, a mixture of linear plasmid DNA , amplified synthetic DNA fragment and T4 DNA polymerase was prepared, as proposed by Thieme et al . ( 2011 ). The mixture was incubated at 25°C for 5 min, and subsequently used for transformation of E. coli Top10 cells (Invitrogen ™ ). Correct constructs were obtained with very high efficiencies (80–95%).

    Article Snippet: After restriction, they were ligated using T4 DNA ligase.

    Techniques: Ligation, Clone Assay, Amplification, Plasmid Preparation, Incubation, Transformation Assay, Construct

    Summary of the Golden GATEway cloning kit. A) Outline of the entire cloning procedure. Eight different entry vectors (pGG-EVx) contain different inserts (colored bars). These inserts are assembled in a predefined order using a Golden Gate reaction into Gateway TM  entry vectors at any position (Threeway Gateway TM  cloning). These can then be assembled to establish a final expression vector in an LR reaction. Compatible overhangs are indicated on each Golden Gate entry vector. B) The principle of Golden Gate cloning is illustrated in the top scheme; the principle of Gateway cloning is illustrated in the bottom scheme. Golden Gate cloning utilizes type II restriction endonucleases to generate compatible overhangs that can be ligated with T4 ligase. The ligation of the two compatible inserts from the entry vectors one and two is illustrated. Gateway cloning relies on recombination of specific att sites using a commercially available enzyme mix (LR Clonase II, Life Technologies).

    Journal: PLoS ONE

    Article Title: Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs

    doi: 10.1371/journal.pone.0076117

    Figure Lengend Snippet: Summary of the Golden GATEway cloning kit. A) Outline of the entire cloning procedure. Eight different entry vectors (pGG-EVx) contain different inserts (colored bars). These inserts are assembled in a predefined order using a Golden Gate reaction into Gateway TM entry vectors at any position (Threeway Gateway TM cloning). These can then be assembled to establish a final expression vector in an LR reaction. Compatible overhangs are indicated on each Golden Gate entry vector. B) The principle of Golden Gate cloning is illustrated in the top scheme; the principle of Gateway cloning is illustrated in the bottom scheme. Golden Gate cloning utilizes type II restriction endonucleases to generate compatible overhangs that can be ligated with T4 ligase. The ligation of the two compatible inserts from the entry vectors one and two is illustrated. Gateway cloning relies on recombination of specific att sites using a commercially available enzyme mix (LR Clonase II, Life Technologies).

    Article Snippet: The 10nM annealed double-stranded oligo dilution was used as an insert in a standard ligation reaction with T4 ligase (5U, Thermo, Fisher)

    Techniques: Clone Assay, Expressing, Plasmid Preparation, Ligation