nebuilder hifi assembly kit  (New England Biolabs)


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    Name:
    NEBuilder HiFi DNA Assembly Cloning Kit
    Description:
    NEBuilder HiFi DNA Assembly Cloning Kit 10 rxns
    Catalog Number:
    e5520s
    Price:
    185
    Size:
    10 rxns
    Category:
    Cloning and Expression Systems
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    Structured Review

    New England Biolabs nebuilder hifi assembly kit
    NEBuilder HiFi DNA Assembly Cloning Kit
    NEBuilder HiFi DNA Assembly Cloning Kit 10 rxns
    https://www.bioz.com/result/nebuilder hifi assembly kit/product/New England Biolabs
    Average 99 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    nebuilder hifi assembly kit - by Bioz Stars, 2020-09
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase
    Article Snippet: .. The assembly of these DNA fragments was performed by using a NEBuilder HiFi DNA Assembly cloning kit (New England BioLabs, Ipswich, MA). .. To construct the PLP knockout mutant, the upstream and downstream regions of PLP were amplified and assembled by using a NEBuilder HiFi DNA Assembly cloning kit.

    Article Title: A new toolkit for gene tagging in Candida albicans containing recyclable markers
    Article Snippet: .. To generate a new pFA series with these recyclable markers for gene disruption, the pFA-CaHIS1 plasmid [ ] was digested with Bam HI and Pme I to release the HIS1 sequence and the pFA backbone was assembled with each of the five modules of the Clox system (LAL , LHL , LUL , URA3-Clox and NAT1-Clox ) using the NEBuilder HiFI DNA Assembly Cloning Kit as described in Materials and Methods. ..

    Article Title: A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging
    Article Snippet: .. The 148 bp PCR amplicon was gel-purified and ligated at a 3:1 ratio to the 415 bp fragment generated by BbsI digest of the pDonor_U6 plasmid (a gift from Andrea Ventura, Addgene plasmid #69312) using the NEBuilder Cloning Kit (New England Biolabs, cat. #E5520S). .. After treatment with Exonuclease RecBCD (New England Biolabs, cat. # M0345L) the column purified DNA plasmid was digested over night at 37 °C with BbsI.

    Article Title: A new toolkit for gene tagging in Candida albicans containing recyclable markers
    Article Snippet: .. Assembly reactions were performed using the NEBuilder HiFI DNA Assembly Cloning Kit (New England Biolabs) following manufacturer’s instructions. .. Design of the primers required for PCR amplification of the different modules was performed with the NEBuilder Assembly tool ( http://nebuilder.neb.com/ ).

    Article Title: A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase
    Article Snippet: .. To construct the PLP knockout mutant, the upstream and downstream regions of PLP were amplified and assembled by using a NEBuilder HiFi DNA Assembly cloning kit. .. BHK-21 cells seeded into 6-well plate were transfected with the full-length cDNA infectious clone of the wild-type virus or its mutant.

    Amplification:

    Article Title: A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging
    Article Snippet: .. The 148 bp PCR amplicon was gel-purified and ligated at a 3:1 ratio to the 415 bp fragment generated by BbsI digest of the pDonor_U6 plasmid (a gift from Andrea Ventura, Addgene plasmid #69312) using the NEBuilder Cloning Kit (New England Biolabs, cat. #E5520S). .. After treatment with Exonuclease RecBCD (New England Biolabs, cat. # M0345L) the column purified DNA plasmid was digested over night at 37 °C with BbsI.

    Article Title: A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase
    Article Snippet: .. To construct the PLP knockout mutant, the upstream and downstream regions of PLP were amplified and assembled by using a NEBuilder HiFi DNA Assembly cloning kit. .. BHK-21 cells seeded into 6-well plate were transfected with the full-length cDNA infectious clone of the wild-type virus or its mutant.

    Mutagenesis:

    Article Title: A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase
    Article Snippet: .. To construct the PLP knockout mutant, the upstream and downstream regions of PLP were amplified and assembled by using a NEBuilder HiFi DNA Assembly cloning kit. .. BHK-21 cells seeded into 6-well plate were transfected with the full-length cDNA infectious clone of the wild-type virus or its mutant.

    Subcloning:

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants
    Article Snippet: .. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. .. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli .

    Generated:

    Article Title: A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging
    Article Snippet: .. The 148 bp PCR amplicon was gel-purified and ligated at a 3:1 ratio to the 415 bp fragment generated by BbsI digest of the pDonor_U6 plasmid (a gift from Andrea Ventura, Addgene plasmid #69312) using the NEBuilder Cloning Kit (New England Biolabs, cat. #E5520S). .. After treatment with Exonuclease RecBCD (New England Biolabs, cat. # M0345L) the column purified DNA plasmid was digested over night at 37 °C with BbsI.

    Construct:

    Article Title: A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase
    Article Snippet: .. To construct the PLP knockout mutant, the upstream and downstream regions of PLP were amplified and assembled by using a NEBuilder HiFi DNA Assembly cloning kit. .. BHK-21 cells seeded into 6-well plate were transfected with the full-length cDNA infectious clone of the wild-type virus or its mutant.

    Sequencing:

    Article Title: A new toolkit for gene tagging in Candida albicans containing recyclable markers
    Article Snippet: .. To generate a new pFA series with these recyclable markers for gene disruption, the pFA-CaHIS1 plasmid [ ] was digested with Bam HI and Pme I to release the HIS1 sequence and the pFA backbone was assembled with each of the five modules of the Clox system (LAL , LHL , LUL , URA3-Clox and NAT1-Clox ) using the NEBuilder HiFI DNA Assembly Cloning Kit as described in Materials and Methods. ..

    Plasmid Purification:

    Article Title: A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase
    Article Snippet: .. To construct the PLP knockout mutant, the upstream and downstream regions of PLP were amplified and assembled by using a NEBuilder HiFi DNA Assembly cloning kit. .. BHK-21 cells seeded into 6-well plate were transfected with the full-length cDNA infectious clone of the wild-type virus or its mutant.

    Two Tailed Test:

    Article Title: Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction
    Article Snippet: .. To assess if there is any statistical significance between the different number of perfect assemblies among genes, the two-tailed Fisher exact test was performed and yielded a p-value of 0.6648 between kanamycin resistance gene and tetracycline resistance gene when using NEBuilder HiFi DNA kit and a p-value < 0.0001 between GFP and kanamycin resistance gene when using the Gibson Assembly kit. .. Therefore, we do not have evidence that the number of perfect assemblies vary with respect to any particular gene assembly using the NEBuilder HiFi DNA Assembly kit, but the results are significantly different for the Gibson Assembly kit at 5% significance level between the two genes tested.

    Significance Assay:

    Article Title: Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction
    Article Snippet: .. To assess if there is any statistical significance between the different number of perfect assemblies among genes, the two-tailed Fisher exact test was performed and yielded a p-value of 0.6648 between kanamycin resistance gene and tetracycline resistance gene when using NEBuilder HiFi DNA kit and a p-value < 0.0001 between GFP and kanamycin resistance gene when using the Gibson Assembly kit. .. Therefore, we do not have evidence that the number of perfect assemblies vary with respect to any particular gene assembly using the NEBuilder HiFi DNA Assembly kit, but the results are significantly different for the Gibson Assembly kit at 5% significance level between the two genes tested.

    Knock-Out:

    Article Title: A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase
    Article Snippet: .. To construct the PLP knockout mutant, the upstream and downstream regions of PLP were amplified and assembled by using a NEBuilder HiFi DNA Assembly cloning kit. .. BHK-21 cells seeded into 6-well plate were transfected with the full-length cDNA infectious clone of the wild-type virus or its mutant.

    Polymerase Chain Reaction:

    Article Title: A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging
    Article Snippet: .. The 148 bp PCR amplicon was gel-purified and ligated at a 3:1 ratio to the 415 bp fragment generated by BbsI digest of the pDonor_U6 plasmid (a gift from Andrea Ventura, Addgene plasmid #69312) using the NEBuilder Cloning Kit (New England Biolabs, cat. #E5520S). .. After treatment with Exonuclease RecBCD (New England Biolabs, cat. # M0345L) the column purified DNA plasmid was digested over night at 37 °C with BbsI.

    Plasmid Preparation:

    Article Title: A new toolkit for gene tagging in Candida albicans containing recyclable markers
    Article Snippet: .. To generate a new pFA series with these recyclable markers for gene disruption, the pFA-CaHIS1 plasmid [ ] was digested with Bam HI and Pme I to release the HIS1 sequence and the pFA backbone was assembled with each of the five modules of the Clox system (LAL , LHL , LUL , URA3-Clox and NAT1-Clox ) using the NEBuilder HiFI DNA Assembly Cloning Kit as described in Materials and Methods. ..

    Article Title: A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging
    Article Snippet: .. The 148 bp PCR amplicon was gel-purified and ligated at a 3:1 ratio to the 415 bp fragment generated by BbsI digest of the pDonor_U6 plasmid (a gift from Andrea Ventura, Addgene plasmid #69312) using the NEBuilder Cloning Kit (New England Biolabs, cat. #E5520S). .. After treatment with Exonuclease RecBCD (New England Biolabs, cat. # M0345L) the column purified DNA plasmid was digested over night at 37 °C with BbsI.

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  • 99
    New England Biolabs nebuilder hifi dna assembly cloning kit
    Construction of the EVG 08/NC_USA/2015 infectious clone and rescue of recombinant viruses. (A) Strategy for assembling the full-length cDNA infectious clone of EVG 08/NC_USA/2015. Two RT-PCR-amplified genomic fragments and a synthesized HDV ribozyme gene were assembled into the pACYC177 vector by using the <t>NEBuilder</t> <t>HiFi</t> <t>DNA</t> assembly method. The CMV promoter sequence was inserted at the 5′ end of the genome. (B) Schematic diagram of the PLP knockout mutant (vPLP-KO). (C) Immunofluorescence detection of recombinant viruses rescued from full-length cDNA clones. BHK-21C cells were initially transfected with plasmid DNA of the full-length cDNA clone pEVG or its mutant. ST cells were subsequently infected with the cloned viruses rescued from BHK cells. The expression of ToV-PLP and the structural protein VP1 was detected by specific MAb 128-28 and MAb 115-5, respectively. The cell nucleus is stained with DAPI. (D) Western blot detection of PLP expression in recombinant virus-infected ST cells. The membrane was probed with ToV-PLP-specific MAb 128-28. The expression of the GAPDH housekeeping gene was detected as a loading control.
    Nebuilder Hifi Dna Assembly Cloning Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nebuilder hifi dna assembly cloning kit/product/New England Biolabs
    Average 99 stars, based on 131 article reviews
    Price from $9.99 to $1999.99
    nebuilder hifi dna assembly cloning kit - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

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    Construction of the EVG 08/NC_USA/2015 infectious clone and rescue of recombinant viruses. (A) Strategy for assembling the full-length cDNA infectious clone of EVG 08/NC_USA/2015. Two RT-PCR-amplified genomic fragments and a synthesized HDV ribozyme gene were assembled into the pACYC177 vector by using the NEBuilder HiFi DNA assembly method. The CMV promoter sequence was inserted at the 5′ end of the genome. (B) Schematic diagram of the PLP knockout mutant (vPLP-KO). (C) Immunofluorescence detection of recombinant viruses rescued from full-length cDNA clones. BHK-21C cells were initially transfected with plasmid DNA of the full-length cDNA clone pEVG or its mutant. ST cells were subsequently infected with the cloned viruses rescued from BHK cells. The expression of ToV-PLP and the structural protein VP1 was detected by specific MAb 128-28 and MAb 115-5, respectively. The cell nucleus is stained with DAPI. (D) Western blot detection of PLP expression in recombinant virus-infected ST cells. The membrane was probed with ToV-PLP-specific MAb 128-28. The expression of the GAPDH housekeeping gene was detected as a loading control.

    Journal: Journal of Virology

    Article Title: A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase

    doi: 10.1128/JVI.00450-17

    Figure Lengend Snippet: Construction of the EVG 08/NC_USA/2015 infectious clone and rescue of recombinant viruses. (A) Strategy for assembling the full-length cDNA infectious clone of EVG 08/NC_USA/2015. Two RT-PCR-amplified genomic fragments and a synthesized HDV ribozyme gene were assembled into the pACYC177 vector by using the NEBuilder HiFi DNA assembly method. The CMV promoter sequence was inserted at the 5′ end of the genome. (B) Schematic diagram of the PLP knockout mutant (vPLP-KO). (C) Immunofluorescence detection of recombinant viruses rescued from full-length cDNA clones. BHK-21C cells were initially transfected with plasmid DNA of the full-length cDNA clone pEVG or its mutant. ST cells were subsequently infected with the cloned viruses rescued from BHK cells. The expression of ToV-PLP and the structural protein VP1 was detected by specific MAb 128-28 and MAb 115-5, respectively. The cell nucleus is stained with DAPI. (D) Western blot detection of PLP expression in recombinant virus-infected ST cells. The membrane was probed with ToV-PLP-specific MAb 128-28. The expression of the GAPDH housekeeping gene was detected as a loading control.

    Article Snippet: To construct the PLP knockout mutant, the upstream and downstream regions of PLP were amplified and assembled by using a NEBuilder HiFi DNA Assembly cloning kit.

    Techniques: Recombinant, Reverse Transcription Polymerase Chain Reaction, Amplification, Synthesized, Plasmid Preparation, Sequencing, Plasmid Purification, Knock-Out, Mutagenesis, Immunofluorescence, Clone Assay, Transfection, Infection, Expressing, Staining, Western Blot

    Modular strategy used for the construction of pFA plasmids with recyclable markers. (A) Construction of pFA deletion plasmids. The five recyclable markers flanked by loxP repeats were amplified with oligonucleotides LXL1 and LXL2, which generated overlapping ends with the pFA-CaHIS1 backbone digested with Bam HI and Pme I. The amplified fragments were assembled with the vector in 5 independent reactions using NEBuilder HiFi DNA Assembly kit. (B) Construction of epitope-tagging plasmids. The different tagging modules (GFPγ, 3xGFPγ, mCherry, 3xHA, 5xmyc or TAP-TAG) were amplified with oligonucleotides that generated overlapping ends with the pFA-CaHIS1 backbone digested with Bam HI (red) and with the 5´end of the recyclable marker module (light blue). The five recyclable markers were independently amplified with oligonucleotides that produced DNA fragments containing overlapping ends with the tagging modules (light blue) and the pFA-CaHIS1 backbone digested with Pme I (yellow). The different modules were assembled with the vector in independent reactions using NEBuilder HiFi DNA Assembly kit.

    Journal: PLoS ONE

    Article Title: A new toolkit for gene tagging in Candida albicans containing recyclable markers

    doi: 10.1371/journal.pone.0219715

    Figure Lengend Snippet: Modular strategy used for the construction of pFA plasmids with recyclable markers. (A) Construction of pFA deletion plasmids. The five recyclable markers flanked by loxP repeats were amplified with oligonucleotides LXL1 and LXL2, which generated overlapping ends with the pFA-CaHIS1 backbone digested with Bam HI and Pme I. The amplified fragments were assembled with the vector in 5 independent reactions using NEBuilder HiFi DNA Assembly kit. (B) Construction of epitope-tagging plasmids. The different tagging modules (GFPγ, 3xGFPγ, mCherry, 3xHA, 5xmyc or TAP-TAG) were amplified with oligonucleotides that generated overlapping ends with the pFA-CaHIS1 backbone digested with Bam HI (red) and with the 5´end of the recyclable marker module (light blue). The five recyclable markers were independently amplified with oligonucleotides that produced DNA fragments containing overlapping ends with the tagging modules (light blue) and the pFA-CaHIS1 backbone digested with Pme I (yellow). The different modules were assembled with the vector in independent reactions using NEBuilder HiFi DNA Assembly kit.

    Article Snippet: Assembly reactions were performed using the NEBuilder HiFI DNA Assembly Cloning Kit (New England Biolabs) following manufacturer’s instructions.

    Techniques: Amplification, Generated, Plasmid Preparation, Marker, Produced

    Agarose gel electrophoresis of the three assembled genes (a) The first lane contains the GFP gene assembled from 27 dsDNA fragments showing up at the expected 757 bp length. The second lane contains the kanamycin resistance gene assembled from 28 dsDNA fragments showing up at the expected 953 bp length. The third lane contains the tetracycline resistance gene assembled from 45 dsDNA fragments at the expected 1254 bp length. All assemblies were performed using Gibson Assembly master mix. (b) The same assemblies performed using the NEBuilder HiFi DNA Assembly master mix. The agarose gel is stained with ethidium bromide.

    Journal: PLoS ONE

    Article Title: Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction

    doi: 10.1371/journal.pone.0145682

    Figure Lengend Snippet: Agarose gel electrophoresis of the three assembled genes (a) The first lane contains the GFP gene assembled from 27 dsDNA fragments showing up at the expected 757 bp length. The second lane contains the kanamycin resistance gene assembled from 28 dsDNA fragments showing up at the expected 953 bp length. The third lane contains the tetracycline resistance gene assembled from 45 dsDNA fragments at the expected 1254 bp length. All assemblies were performed using Gibson Assembly master mix. (b) The same assemblies performed using the NEBuilder HiFi DNA Assembly master mix. The agarose gel is stained with ethidium bromide.

    Article Snippet: Thus, our method can potentially be useful for the construction of other genes using the NEBuilder HiFi Assembly kit.

    Techniques: Agarose Gel Electrophoresis, Staining