golden gate assembly  (New England Biolabs)


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    Name:
    NEB Golden Gate Assembly Mix
    Description:
    The NEB Golden Gate Assembly Kit BsaI HFv2 contains an optimized mix of BsaI HFv2 and T4 DNA Ligase Together these enzymes along with an optimal buffer can direct the assembly of multiple inserts modules using the Golden Gate approach Also provided is the pGGA destination plasmid which provides a backbone for your assembly features convenient restriction enzyme sites for subcloning and has T7 SP6 promoter sequences to enable in vitro transcription
    Catalog Number:
    e1600s
    Price:
    400
    Size:
    100 rxns
    Category:
    DNA Fragment Analysis Kits
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    Structured Review

    New England Biolabs golden gate assembly
    NEB Golden Gate Assembly Mix
    The NEB Golden Gate Assembly Kit BsaI HFv2 contains an optimized mix of BsaI HFv2 and T4 DNA Ligase Together these enzymes along with an optimal buffer can direct the assembly of multiple inserts modules using the Golden Gate approach Also provided is the pGGA destination plasmid which provides a backbone for your assembly features convenient restriction enzyme sites for subcloning and has T7 SP6 promoter sequences to enable in vitro transcription
    https://www.bioz.com/result/golden gate assembly/product/New England Biolabs
    Average 99 stars, based on 6 article reviews
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    golden gate assembly - by Bioz Stars, 2020-09
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    Polymerase Chain Reaction:

    Article Title: Tunable genetic devices through simultaneous control of transcription and translation
    Article Snippet: .. Removal of RiboJ from the TES (pVB001) and NOT gate (pVB002) was achieved by PCR of the relevant design using primers F_RiboJ_Rem and R_RiboJ_Rem ( Supplementary Table 5 ) and subsequent circularization by standard Golden Gate assembly (New England Biolabs, E1601S) to create the plasmids pVB003 and pVB004, respectively. .. The plasmid used to boost tuner sRNA levels (pVB005) was fully synthesized (GeneArt, Thermo Fisher Scientific).

    Article Title: Large scale active-learning-guided exploration for in vitro protein production optimization
    Article Snippet: .. The PCR amplifications was followed by a golden gate assembly using BsaI and T4 ligase (New England Biolab) and transformed into chemically competent E. coli top10. .. Plasmid preparation We noticed with preliminary experiments that the same cell-compositions gave different results when we used plasmid DNA from miniprep done on different days using the same kit.

    Article Title: Tunable genetic devices through simultaneous control of transcription and translation
    Article Snippet: .. Removal of RiboJ from the TES (pVB001) and NOT gate (pVB002) was achieved by PCR of the relevant design using primers F_RiboJ_Rem and R_RiboJ_Rem (Supplementary Table ) and subsequent circularization by standard Golden Gate assembly (New England Biolabs, E1601S) to create the plasmids pVB003 and pVB004, respectively. .. The plasmid used to boost tuner sRNA levels (pVB005) was fully synthesized (GeneArt, Thermo Fisher Scientific).

    Clone Assay:

    Article Title: A single CRISPR base editor to induce simultaneous C-to-T and A-to-G mutations
    Article Snippet: .. The annealed spacer inserts were then ligated into a pU6-gRNA cloning backbone (pSI-356) by Golden Gate Assembly using BsmBI (NEB) and T4 DNA ligase (NEB). .. Lentiviral base editing reporter plasmidsTo construct the lentiviral C-to-T base editing reporter plasmid (pLV-SI-112), the reporter cassette was amplified from pLV-eGFP (Addgene 36083) using 112-V4-BC2-FW/EGFP-BamH1-RV and cloned into EcoRI and BamHI sites of pLVSIN-CMV-Puro backbone vector (Takara) using T4 DNA ligase (NEB).

    Transformation Assay:

    Article Title: Large scale active-learning-guided exploration for in vitro protein production optimization
    Article Snippet: .. The PCR amplifications was followed by a golden gate assembly using BsaI and T4 ligase (New England Biolab) and transformed into chemically competent E. coli top10. .. Plasmid preparation We noticed with preliminary experiments that the same cell-compositions gave different results when we used plasmid DNA from miniprep done on different days using the same kit.

    Plasmid Preparation:

    Article Title: CTCF Promotes Long-range Enhancer-promoter Interactions and Lineage-specific Gene Expression in Mammalian Cells
    Article Snippet: .. The three gRNA-expressing cassettes were incorporated into one single plasmid using Golden Gate assembly. .. The donor vector (mCTCF24 -AID-donor-Neo) was constructed using PCR and Gibson Assembly Cloning kit (New England Biolabs).

    Expressing:

    Article Title: Repurposing the yeast peroxisome to compartmentalize a toxic enzyme enables improved (S)-reticuline production
    Article Snippet: .. Yeast strain construction Yeast expression vectors were built using Golden Gate Assembly as described in the YTK system . .. Integration into the yeast genome via homologous recombination at the URA3 or LEU2 locus was achieved by transformation of linearized plasmids (NotI digestion, NEB) whereas replicating CEN6/ARS4 or 2µ plasmids were transformed directly into yeast without pre-digestion with NotI.

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    New England Biolabs bsa i
    Assembly strategy and hierarchical backbone levels of the cloning systems GoldenMOCS and Golden Pi CS. In the microorganism-independent general platform GoldenMOCS, DNA products (synthetic DNA, PCR products or oligonucleotides) are integrated into BB1 by a <t>Bsa</t> I Golden Gate Assembly and fusion sites Fs1, Fs2, Fs3 and Fs4. Fusion sites are indicated as colored boxes with corresponding fusion site number or letter. Basic genetic elements contained in backbone 1 (BB1) can be assembled in recipient BB2 by performing a Bpi I GGA reaction. The transcription units in BB2 are further used for Bsa I assembly into multigene BB3 constructs. Single transcription units can be obtained by direct Bpi I assembly into recipient BB3 with fusion sites Fs1-Fs4. Fusion sites determine module and transcription unit positions in assembled constructs. Thereby, fusion sites Fs1 to Fs4 are used to construct single expression cassettes in BB2 and are required between promoter (Fs1-Fs2), CDS (Fs2-Fs3) and terminator (Fs3-Fs4). Fusion sites FsA to FsI are designed to construct BB3 plasmids and separate the different expression cassettes from each other. The FSs are almost randomly chosen sequences and only FS2 has a special function, because it includes the start codon ATG. Golden Pi CS additionally includes module-containing BB1s specific for P. pastoris : 20 promoters, 1 reporter gene (eGFP) and 10 transcription terminators, and recipient BB3 vectors containing different integration loci for stable genome integration in P. pastoris and suitable resistance cassettes (Additional file 2 )
    Bsa I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    New England Biolabs golden gate assembly gga reactions
    Golden Transformation can achieve high genome editing rates. Transformation of two different cassettes constructed in 3-part or 5-part Golden Gate assembly <t>(GGA)</t> reactions were compared by counting colonies obtained on LB-Kan versus LB agar. Transformations of a purified <t>DNA</t> sample constructed by overlap-extension PCR (OE-PCR) at a concentration that corresponds to 100% efficient assembly of the 3-part GGA reaction and this OE-PCR sample with GGA buffer added to it were included for comparison. The effects of “short” (1 hr) or “long” (5 hr) GGA thermocycling programs, using T4 versus T7 ligase in GGA reactions, and performing “puddle” transformations that concentrate cells and DNA by combining them on a filter placed on an agar surface rather than in a liquid culture were also tested.
    Golden Gate Assembly Gga Reactions, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs restriction enzymes bsa i
    Extension of the Plant Golden Gate MoClo Assembly Standard for cyanobacterial transformation. Assembly relies on one of two Type IIS restriction endonuclease enzymes ( <t>Bsa</t> I or Bpi for the full list and maps of level T acceptor vectors.
    Restriction Enzymes Bsa I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Assembly strategy and hierarchical backbone levels of the cloning systems GoldenMOCS and Golden Pi CS. In the microorganism-independent general platform GoldenMOCS, DNA products (synthetic DNA, PCR products or oligonucleotides) are integrated into BB1 by a Bsa I Golden Gate Assembly and fusion sites Fs1, Fs2, Fs3 and Fs4. Fusion sites are indicated as colored boxes with corresponding fusion site number or letter. Basic genetic elements contained in backbone 1 (BB1) can be assembled in recipient BB2 by performing a Bpi I GGA reaction. The transcription units in BB2 are further used for Bsa I assembly into multigene BB3 constructs. Single transcription units can be obtained by direct Bpi I assembly into recipient BB3 with fusion sites Fs1-Fs4. Fusion sites determine module and transcription unit positions in assembled constructs. Thereby, fusion sites Fs1 to Fs4 are used to construct single expression cassettes in BB2 and are required between promoter (Fs1-Fs2), CDS (Fs2-Fs3) and terminator (Fs3-Fs4). Fusion sites FsA to FsI are designed to construct BB3 plasmids and separate the different expression cassettes from each other. The FSs are almost randomly chosen sequences and only FS2 has a special function, because it includes the start codon ATG. Golden Pi CS additionally includes module-containing BB1s specific for P. pastoris : 20 promoters, 1 reporter gene (eGFP) and 10 transcription terminators, and recipient BB3 vectors containing different integration loci for stable genome integration in P. pastoris and suitable resistance cassettes (Additional file 2 )

    Journal: BMC Systems Biology

    Article Title: GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris

    doi: 10.1186/s12918-017-0492-3

    Figure Lengend Snippet: Assembly strategy and hierarchical backbone levels of the cloning systems GoldenMOCS and Golden Pi CS. In the microorganism-independent general platform GoldenMOCS, DNA products (synthetic DNA, PCR products or oligonucleotides) are integrated into BB1 by a Bsa I Golden Gate Assembly and fusion sites Fs1, Fs2, Fs3 and Fs4. Fusion sites are indicated as colored boxes with corresponding fusion site number or letter. Basic genetic elements contained in backbone 1 (BB1) can be assembled in recipient BB2 by performing a Bpi I GGA reaction. The transcription units in BB2 are further used for Bsa I assembly into multigene BB3 constructs. Single transcription units can be obtained by direct Bpi I assembly into recipient BB3 with fusion sites Fs1-Fs4. Fusion sites determine module and transcription unit positions in assembled constructs. Thereby, fusion sites Fs1 to Fs4 are used to construct single expression cassettes in BB2 and are required between promoter (Fs1-Fs2), CDS (Fs2-Fs3) and terminator (Fs3-Fs4). Fusion sites FsA to FsI are designed to construct BB3 plasmids and separate the different expression cassettes from each other. The FSs are almost randomly chosen sequences and only FS2 has a special function, because it includes the start codon ATG. Golden Pi CS additionally includes module-containing BB1s specific for P. pastoris : 20 promoters, 1 reporter gene (eGFP) and 10 transcription terminators, and recipient BB3 vectors containing different integration loci for stable genome integration in P. pastoris and suitable resistance cassettes (Additional file 2 )

    Article Snippet: Golden Gate assembly reaction One μL Bsa I or Bpi I (10 U), 40 U T4 Ligase (0.1 μL), 2 μL CutSmart™ Buffer (10×, NEB), 2 μL ATP (10 mM, NEB) and 40 nM dilutions of PCR fragments and/or carrier and recipient backbone were diluted in 20 μL total volume and incubated as follows: 8 to 50 cycles (depending on insert number) of each 2 min at 37 °C and 16 °C, followed by 10 min at 37 °C, 30 min at 55 °C and 10 min at 80 °C (final ligation, digestion and heat inactivation).

    Techniques: Clone Assay, Polymerase Chain Reaction, Construct, Expressing

    Golden Transformation can achieve high genome editing rates. Transformation of two different cassettes constructed in 3-part or 5-part Golden Gate assembly (GGA) reactions were compared by counting colonies obtained on LB-Kan versus LB agar. Transformations of a purified DNA sample constructed by overlap-extension PCR (OE-PCR) at a concentration that corresponds to 100% efficient assembly of the 3-part GGA reaction and this OE-PCR sample with GGA buffer added to it were included for comparison. The effects of “short” (1 hr) or “long” (5 hr) GGA thermocycling programs, using T4 versus T7 ligase in GGA reactions, and performing “puddle” transformations that concentrate cells and DNA by combining them on a filter placed on an agar surface rather than in a liquid culture were also tested.

    Journal: bioRxiv

    Article Title: Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

    doi: 10.1101/754242

    Figure Lengend Snippet: Golden Transformation can achieve high genome editing rates. Transformation of two different cassettes constructed in 3-part or 5-part Golden Gate assembly (GGA) reactions were compared by counting colonies obtained on LB-Kan versus LB agar. Transformations of a purified DNA sample constructed by overlap-extension PCR (OE-PCR) at a concentration that corresponds to 100% efficient assembly of the 3-part GGA reaction and this OE-PCR sample with GGA buffer added to it were included for comparison. The effects of “short” (1 hr) or “long” (5 hr) GGA thermocycling programs, using T4 versus T7 ligase in GGA reactions, and performing “puddle” transformations that concentrate cells and DNA by combining them on a filter placed on an agar surface rather than in a liquid culture were also tested.

    Article Snippet: Golden Gate assembly DNA fragments for transformation were constructed using Golden Gate Assembly (GGA) reactions containing 1 U/µl BsaI-HF or 0.5 U/µl BsmBI and 150 U/µl of either T7 or T4 DNA ligase in T4 DNA ligase buffer (New England Biolabs) ( , ).

    Techniques: Transformation Assay, Construct, Purification, Polymerase Chain Reaction, Overlap Extension Polymerase Chain Reaction, Concentration Assay

    Golden Transformation method for ADP1 genome engineering. Two PCR reactions are performed to create upstream (U) and downstream (D) genomic target flanks with added terminal BsaI and BsmBI type IIS restriction sites as depicted. The two PCR products can then be combined via BsaI Golden Gate assembly (GGA) with the selection cassette to form a replacement DNA or combined with one another and optionally with additional genetic parts (not shown) via BsmBI GGA to form a rescue cassette. The positive-negative selection cassette ( tdk - kanR ) is maintained on the high-copy pBTK622 plasmid that has an origin that does not replicate in A. baylyi . The first GGA reaction is added to an A. baylyi culture and then plated on LB-Kan to select for transformants with the replacement cassette integrated into the genome. Then, transformation of the second assembly reaction with counterselection on LB-AZT is used to move the unmarked deletions/additions encoded on the rescue cassette into the genome.

    Journal: bioRxiv

    Article Title: Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

    doi: 10.1101/754242

    Figure Lengend Snippet: Golden Transformation method for ADP1 genome engineering. Two PCR reactions are performed to create upstream (U) and downstream (D) genomic target flanks with added terminal BsaI and BsmBI type IIS restriction sites as depicted. The two PCR products can then be combined via BsaI Golden Gate assembly (GGA) with the selection cassette to form a replacement DNA or combined with one another and optionally with additional genetic parts (not shown) via BsmBI GGA to form a rescue cassette. The positive-negative selection cassette ( tdk - kanR ) is maintained on the high-copy pBTK622 plasmid that has an origin that does not replicate in A. baylyi . The first GGA reaction is added to an A. baylyi culture and then plated on LB-Kan to select for transformants with the replacement cassette integrated into the genome. Then, transformation of the second assembly reaction with counterselection on LB-AZT is used to move the unmarked deletions/additions encoded on the rescue cassette into the genome.

    Article Snippet: Golden Gate assembly DNA fragments for transformation were constructed using Golden Gate Assembly (GGA) reactions containing 1 U/µl BsaI-HF or 0.5 U/µl BsmBI and 150 U/µl of either T7 or T4 DNA ligase in T4 DNA ligase buffer (New England Biolabs) ( , ).

    Techniques: Transformation Assay, Polymerase Chain Reaction, Selection, Plasmid Preparation

    Extension of the Plant Golden Gate MoClo Assembly Standard for cyanobacterial transformation. Assembly relies on one of two Type IIS restriction endonuclease enzymes ( Bsa I or Bpi for the full list and maps of level T acceptor vectors.

    Journal: Plant Physiology

    Article Title: CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax 1CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax 1 [OPEN]

    doi: 10.1104/pp.18.01401

    Figure Lengend Snippet: Extension of the Plant Golden Gate MoClo Assembly Standard for cyanobacterial transformation. Assembly relies on one of two Type IIS restriction endonuclease enzymes ( Bsa I or Bpi for the full list and maps of level T acceptor vectors.

    Article Snippet: Golden Gate assembly reactions were performed with restriction enzymes Bsa I (New England Biolabs) or Bpi for detailed protocols).

    Techniques: Transformation Assay