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Product Information
Gibson Assembly was developed by Dr. Daniel Gibson and his colleagues at the J. Craig Venter Institute and licensed to NEB by Synthetic Genomics, Inc. It allows for successful assembly of multiple DNA fragments, regardless of fragment length or end compatibility. It has been rapidly adopted by the synthetic biology community due to its ease-of-use, flexibility and suitability for large DNA constructs.
Gibson Assembly efficiently joins multiple overlapping DNA fragments in a single-tube isothermal reaction (1,2). The Gibson Assembly Master Mix includes three different enzymatic activities that perform in a single buffer:
- The exonuclease creates single-stranded 3´ overhangs that facilitate the annealing of fragments that share complementarity at one end (overlap region).
- The proprietary DNA polymerase fills in gaps within each annealed fragment.
- The DNA ligase seals nicks in the assembled DNA.
The end result is a double-stranded fully sealed DNA molecule that can serve as template for PCR, RCA or a variety of other molecular biology applications, including direct transformation. The method has been successfully used by Gibson’s group and others to assemble oligonucleotides, DNA with varied overlaps (15–80 bp) and fragments hundreds of kilobases long (1–2).
To help select the best DNA assembly method for your needs, please use our Synthetic Biology/DNA Assembly Selection Chart.
For help designing primers, please view our primer design video.
Overview of the Gibson Assembly Cloning Method
10 μl of 2X Gibson Assembly Master Mix was incubated with 6 fragments (5 fragments of 400 bp and one of 2,780 bp, with 40 bp overlap, 0.05 pmol each) in a final volume of 20 μl at 50°C for 60 minutes. NEB 5-alpha Competent E. coli (NEB #C2987) were transformed with 2 μl of the master mix/fragment mixture using the transformation protocol on page 12. Greater than 100 white colonies were observed when 1/10 of the outgrowth was spread on an ampicillin plate with IPTG/Xgal and incubated overnight.
Specification:
Overview of Gibson Assembly Cloning Kit Protocol:
- Design primers to amplify fragments (and/or vector) with appropriate overlaps
- PCR amplify fragments using a high-fidelity DNA polymerase.
- Prepare linearized vector by PCR amplification using a high-fidelity DNA polymerase or by restriction digestion.
- Confirm and determine concentration of fragments and linearized vector using agarose gel electrophoresis, a NanoDrop™ instrument or other method.
- Add fragments and linearized vector to Gibson Assembly Master Mix and incubate at 50°C for 15 minutes to 1 hour, depending on number of fragments being assembled.
- Transform into NEB 5-alpha Competent E. coli (provided) or use directly in other applications.
- This product is related to the following categories:
- DNA Assembly, Cloning and Mutagenesis Kits Products
- This product can be used in the following applications:
- Gibson Assembly®
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Protocols, Manuals & Usage
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Citations & Technical Literature
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Product Citation Tool
Additional Citations
- Ikmi A, Gaertner B, Seidel C, Srivastava M, Zeitlinger J, Gibson MC (2014) Molecular evolution of the Yap/Yorkie proto-oncogene and elucidation of its core transcriptional program Mol Biol Evol; 31(6), 1375-90. PubMedID: 24509725, DOI: 10.1093/molbev/msu071
- Horii T, Arai Y, Yamazaki M, Morita S, Kimura M, Itoh M, Abe Y, Hatada I (2014) Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering Sci Rep; 4, 4513. PubMedID: 24675426, DOI: 10.1038/srep04513
- Schöner TA, Fuchs SW, Reinhold-Hurek B, Bode HB (2014) Identification and Biosynthesis of a Novel Xanthomonadin-Dialkylresorcinol-Hybrid from Azoarcus sp. BH72 PLoS One; 9(3), e90922. PubMedID: 24618669, DOI: 10.1371/journal.pone.0090922
- Ikmi A, Gaertner B, Seidel C, Srivastava M, Zeitlinger J, Gibson MC (2014) Molecular evolution of the Yap/Yorkie proto-oncogene and elucidation of its core transcriptional program Mol Biol Evol; 31(6), 1375-90. PubMedID: 24509725, DOI: 10.1093/molbev/msu071
- Li Y, Thompson CM, Lipsitch M (2014) A Modified Janus Cassette (Sweet Janus) to Improve Allelic Replacement Efficiency by High-Stringency Negative Selection in Streptococcus pneumoniae PLoS One; 9(6), e100510. PubMedID: 24959661, DOI: 10.1371/journal.pone.0100510
- Ng S, Ivanova A, Duncan O, Law SR, Van Aken O, De Clercq I, Wang Y, Carrie C, Xu L, Kmiec B, Walker H, Van Breusegem F, Whelan J, Giraud E (2013) A membrane-bound NAC transcription factor, ANAC017, mediates mitochondrial retrograde signaling in Arabidopsis Plant Cell; 25(9), 3450-71. PubMedID: 24045017, DOI: 10.1105/tpc.113.113985
- Guye P, Li Y, Wroblewska L, Duportet X, Weiss R (2013) Rapid, modular and reliable construction of complex mammalian gene circuits Nucleic Acids Res; 41(16), e156. PubMedID: 23847100, DOI: 10.1093/nar/gkt605
- Ramirez-Peralta A, Gupta S, Butzin XY, Setlow B, Korza G, Leyva-Vazquez MA, Christie G, Setlow P (2013) Identification of new proteins that modulate the germination of spores of bacillus species J Bacteriol; 195(13), 3009-21. PubMedID: 23625846, DOI: 10.1128/JB.00257-13
- Singh R, Low ET, Ooi LC, Ong-Abdullah M, Ting NC, Nagappan J, Nookiah R, Amiruddin MD, Rosli R, Manaf MA, Chan KL, Halim MA, Azizi N, Lakey N, Smith SW, Budiman MA, Hogan M, Bacher B, Van Brunt A, Wang C, Ordway JM, Sambanthamurthi R, Martienssen RA (2013) The oil palm SHELL gene controls oil yield and encodes a homologue of SEEDSTICK Nature; 500(7462), 340-4. PubMedID: 23883930, DOI: 10.1038/nature12356.
- Chen C, Fenk LA, de Bono M (2013) Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination Nucleic Acids Res; 41(20), e193. PubMedID: 24013562, DOI: 10.1093/nar/gkt805
- DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems Nucleic Acids Res; 41(7), 4336-43. PubMedID: 23460208, DOI: 10.1093/nar/gkt135
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Featured Videos
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Introduction to Gibson Assembly®
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Primer Design and Fragment Assembly Using NEBuilder HiFi DNA Assembly® or Gibson Assembly®
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Gibson Assembly Workflow
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NEBUILDER® Assembly Tool 2.0 What’s New?
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NEBUILDER® Assembly Tool 2.0 Fragments Amplified by PCR
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NEBUILDER® Assembly Tool 2.0 Restriction Enzyme Digest
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