golden gate assembly kit  (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
    Buy from Supplier


    Structured Review

    New England Biolabs golden gate assembly kit
    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 kit/product/New England Biolabs
    Average 99 stars, based on 1658 article reviews
    Price from $9.99 to $1999.99
    golden gate assembly kit - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Porcupine: Rapid and robust tagging of physical objects using nanopore-orthogonal DNA strands"

    Article Title: Porcupine: Rapid and robust tagging of physical objects using nanopore-orthogonal DNA strands

    Journal: bioRxiv

    doi: 10.1101/2020.03.06.981514

    Molbit design scheme. (a) Molecular bit (molbit) structure. The molbit sequence is attached to a spacer sequence via Golden Gate assembly to achieve a minimum length for sequencing and provide an additional encoding channel. Since the sequencing adapter is attached to both ends, the strand can be sensed from either direction. (b) The letters “UW” depicted visually in nanopore raw data (as opposed to encoded in the sequence contents). From top to bottom, the shown sequence was simulated using Scrappie and sequenced on the ONT Min-ION, demonstrating the viability of using simulations for designing intentional, arbitrary raw signal shapes. (c) Evolutionary model workflow. Each round of evolution begins with a set of sequences, their simulated squiggles, and pairwise Dynamic Time Warping (DTW) distances. The sequence order is randomized, and sequences are mutated one at a time, verifying DTW improvement (minimum and mean) after each attempt. (d) Dynamic time warping (DTW) scores before (left) and after (right) 31 iterations of the evolutionary model. After initialization, the minimum DTW similarity was 2.9 (mean 4.2 +/- 0.4), and after evolution the minimum was 4.2 (mean 5.8 +/- 0.8).
    Figure Legend Snippet: Molbit design scheme. (a) Molecular bit (molbit) structure. The molbit sequence is attached to a spacer sequence via Golden Gate assembly to achieve a minimum length for sequencing and provide an additional encoding channel. Since the sequencing adapter is attached to both ends, the strand can be sensed from either direction. (b) The letters “UW” depicted visually in nanopore raw data (as opposed to encoded in the sequence contents). From top to bottom, the shown sequence was simulated using Scrappie and sequenced on the ONT Min-ION, demonstrating the viability of using simulations for designing intentional, arbitrary raw signal shapes. (c) Evolutionary model workflow. Each round of evolution begins with a set of sequences, their simulated squiggles, and pairwise Dynamic Time Warping (DTW) distances. The sequence order is randomized, and sequences are mutated one at a time, verifying DTW improvement (minimum and mean) after each attempt. (d) Dynamic time warping (DTW) scores before (left) and after (right) 31 iterations of the evolutionary model. After initialization, the minimum DTW similarity was 2.9 (mean 4.2 +/- 0.4), and after evolution the minimum was 4.2 (mean 5.8 +/- 0.8).

    Techniques Used: Sequencing

    Related Articles

    Transduction:

    Article Title: Dissecting super-enhancer hierarchy based on chromatin interactions
    Article Snippet: .. The annealed oligos were cloned into pLV-hU6-sgRNA-hUbC-dCas9-KRAB-T2a-Puro vector (Addgene ID: 71236) using a Golden Gate Assembly strategy including: 100 ng of circular pLV plasmid, 0.2 μM annealed oligos, 2.1 buffer (1×) (New England Biolabs), 20 U of BsmBI restriction enzyme, 0.2 mM ATP, 0.1 mg/ml BSA, and 750 U of T4 DNA ligase (New England Biolabs) with the cycling parameters of 20 cycles of 37 °C for 5 min, 20 °C for 5 min; followed by 80 °C incubation for 20 min. Then K562 cells were transduced with lentivirus to stably express dCa s9-KRAB and sgRNA. .. Briefly, sequence-specific sgRNAs for site-specific interference of genomic targets were designed following described guidelines, and sequences were selected to minimize off-target effect based on publicly available filtering tools ( http://crispr.mit.edu/ ).

    Clone Assay:

    Article Title: Dissecting super-enhancer hierarchy based on chromatin interactions
    Article Snippet: .. The annealed oligos were cloned into pLV-hU6-sgRNA-hUbC-dCas9-KRAB-T2a-Puro vector (Addgene ID: 71236) using a Golden Gate Assembly strategy including: 100 ng of circular pLV plasmid, 0.2 μM annealed oligos, 2.1 buffer (1×) (New England Biolabs), 20 U of BsmBI restriction enzyme, 0.2 mM ATP, 0.1 mg/ml BSA, and 750 U of T4 DNA ligase (New England Biolabs) with the cycling parameters of 20 cycles of 37 °C for 5 min, 20 °C for 5 min; followed by 80 °C incubation for 20 min. Then K562 cells were transduced with lentivirus to stably express dCa s9-KRAB and sgRNA. .. Briefly, sequence-specific sgRNAs for site-specific interference of genomic targets were designed following described guidelines, and sequences were selected to minimize off-target effect based on publicly available filtering tools ( http://crispr.mit.edu/ ).

    Stable Transfection:

    Article Title: Dissecting super-enhancer hierarchy based on chromatin interactions
    Article Snippet: .. The annealed oligos were cloned into pLV-hU6-sgRNA-hUbC-dCas9-KRAB-T2a-Puro vector (Addgene ID: 71236) using a Golden Gate Assembly strategy including: 100 ng of circular pLV plasmid, 0.2 μM annealed oligos, 2.1 buffer (1×) (New England Biolabs), 20 U of BsmBI restriction enzyme, 0.2 mM ATP, 0.1 mg/ml BSA, and 750 U of T4 DNA ligase (New England Biolabs) with the cycling parameters of 20 cycles of 37 °C for 5 min, 20 °C for 5 min; followed by 80 °C incubation for 20 min. Then K562 cells were transduced with lentivirus to stably express dCa s9-KRAB and sgRNA. .. Briefly, sequence-specific sgRNAs for site-specific interference of genomic targets were designed following described guidelines, and sequences were selected to minimize off-target effect based on publicly available filtering tools ( http://crispr.mit.edu/ ).

    Mutagenesis:

    Article Title: Functional Plasticity and Evolutionary Adaptation of Allosteric Regulation
    Article Snippet: .. We assembled mutant libraries by combining the linearized sensor backbone with each oligo subpool at a molar ratio of 1:5 using Golden Gate Assembly Kit (New England Biolabs; 37 °C for 5 min and 60 °C for 5 min, repeated 30x). .. Reactions were dialyzed with water on silica membranes (0.025 μm pores) for 1 hour before transformed into DH10β cells (New England Biolab).

    Incubation:

    Article Title: Dissecting super-enhancer hierarchy based on chromatin interactions
    Article Snippet: .. The annealed oligos were cloned into pLV-hU6-sgRNA-hUbC-dCas9-KRAB-T2a-Puro vector (Addgene ID: 71236) using a Golden Gate Assembly strategy including: 100 ng of circular pLV plasmid, 0.2 μM annealed oligos, 2.1 buffer (1×) (New England Biolabs), 20 U of BsmBI restriction enzyme, 0.2 mM ATP, 0.1 mg/ml BSA, and 750 U of T4 DNA ligase (New England Biolabs) with the cycling parameters of 20 cycles of 37 °C for 5 min, 20 °C for 5 min; followed by 80 °C incubation for 20 min. Then K562 cells were transduced with lentivirus to stably express dCa s9-KRAB and sgRNA. .. Briefly, sequence-specific sgRNAs for site-specific interference of genomic targets were designed following described guidelines, and sequences were selected to minimize off-target effect based on publicly available filtering tools ( http://crispr.mit.edu/ ).

    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.

    Polymerase Chain Reaction:

    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).

    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).

    Article Title: Dissecting super-enhancer hierarchy based on chromatin interactions
    Article Snippet: .. The annealed oligos were cloned into pLV-hU6-sgRNA-hUbC-dCas9-KRAB-T2a-Puro vector (Addgene ID: 71236) using a Golden Gate Assembly strategy including: 100 ng of circular pLV plasmid, 0.2 μM annealed oligos, 2.1 buffer (1×) (New England Biolabs), 20 U of BsmBI restriction enzyme, 0.2 mM ATP, 0.1 mg/ml BSA, and 750 U of T4 DNA ligase (New England Biolabs) with the cycling parameters of 20 cycles of 37 °C for 5 min, 20 °C for 5 min; followed by 80 °C incubation for 20 min. Then K562 cells were transduced with lentivirus to stably express dCa s9-KRAB and sgRNA. .. Briefly, sequence-specific sgRNAs for site-specific interference of genomic targets were designed following described guidelines, and sequences were selected to minimize off-target effect based on publicly available filtering tools ( http://crispr.mit.edu/ ).

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    New England Biolabs golden gate assembly kit
    Molbit design scheme. (a) Molecular bit (molbit) structure. The molbit sequence is attached to a spacer sequence via <t>Golden</t> <t>Gate</t> <t>assembly</t> to achieve a minimum length for sequencing and provide an additional encoding channel. Since the sequencing adapter is attached to both ends, the strand can be sensed from either direction. (b) The letters “UW” depicted visually in nanopore raw data (as opposed to encoded in the sequence contents). From top to bottom, the shown sequence was simulated using Scrappie and sequenced on the ONT Min-ION, demonstrating the viability of using simulations for designing intentional, arbitrary raw signal shapes. (c) Evolutionary model workflow. Each round of evolution begins with a set of sequences, their simulated squiggles, and pairwise Dynamic Time Warping (DTW) distances. The sequence order is randomized, and sequences are mutated one at a time, verifying DTW improvement (minimum and mean) after each attempt. (d) Dynamic time warping (DTW) scores before (left) and after (right) 31 iterations of the evolutionary model. After initialization, the minimum DTW similarity was 2.9 (mean 4.2 +/- 0.4), and after evolution the minimum was 4.2 (mean 5.8 +/- 0.8).
    Golden Gate Assembly Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/golden gate assembly kit/product/New England Biolabs
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    golden gate assembly kit - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

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    Molbit design scheme. (a) Molecular bit (molbit) structure. The molbit sequence is attached to a spacer sequence via Golden Gate assembly to achieve a minimum length for sequencing and provide an additional encoding channel. Since the sequencing adapter is attached to both ends, the strand can be sensed from either direction. (b) The letters “UW” depicted visually in nanopore raw data (as opposed to encoded in the sequence contents). From top to bottom, the shown sequence was simulated using Scrappie and sequenced on the ONT Min-ION, demonstrating the viability of using simulations for designing intentional, arbitrary raw signal shapes. (c) Evolutionary model workflow. Each round of evolution begins with a set of sequences, their simulated squiggles, and pairwise Dynamic Time Warping (DTW) distances. The sequence order is randomized, and sequences are mutated one at a time, verifying DTW improvement (minimum and mean) after each attempt. (d) Dynamic time warping (DTW) scores before (left) and after (right) 31 iterations of the evolutionary model. After initialization, the minimum DTW similarity was 2.9 (mean 4.2 +/- 0.4), and after evolution the minimum was 4.2 (mean 5.8 +/- 0.8).

    Journal: bioRxiv

    Article Title: Porcupine: Rapid and robust tagging of physical objects using nanopore-orthogonal DNA strands

    doi: 10.1101/2020.03.06.981514

    Figure Lengend Snippet: Molbit design scheme. (a) Molecular bit (molbit) structure. The molbit sequence is attached to a spacer sequence via Golden Gate assembly to achieve a minimum length for sequencing and provide an additional encoding channel. Since the sequencing adapter is attached to both ends, the strand can be sensed from either direction. (b) The letters “UW” depicted visually in nanopore raw data (as opposed to encoded in the sequence contents). From top to bottom, the shown sequence was simulated using Scrappie and sequenced on the ONT Min-ION, demonstrating the viability of using simulations for designing intentional, arbitrary raw signal shapes. (c) Evolutionary model workflow. Each round of evolution begins with a set of sequences, their simulated squiggles, and pairwise Dynamic Time Warping (DTW) distances. The sequence order is randomized, and sequences are mutated one at a time, verifying DTW improvement (minimum and mean) after each attempt. (d) Dynamic time warping (DTW) scores before (left) and after (right) 31 iterations of the evolutionary model. After initialization, the minimum DTW similarity was 2.9 (mean 4.2 +/- 0.4), and after evolution the minimum was 4.2 (mean 5.8 +/- 0.8).

    Article Snippet: To assemble the molbits, 600 ng of the desired annealed barcodes (at equimolar concentrations) and 600 ng of the spacer were ligated together using NEB’s Golden Gate Assembly Kit.

    Techniques: Sequencing