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  • Gibson Assembly® Cloning Kit

    Description

      
    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.

    Overview of the Gibson Assembly Cloning Method

    Overview of the Gibson Assembly Cloning Method


    Specification:

    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.

    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.

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Gibson Assembly® Master Mix-20*2X
    NEBuilder® Positive Control-202X
    NEB 5-alpha Competent E. coli (High Efficiency)-80
    SOC Outgrowth Medium41X
    pUC19 Transformation Control Plasmid-200.05 ng/μl

    * Reagents' Storage Notes

    • Gibson Assembly® Master Mix: Store at -20°C. Thaw, vortex thoroughly before use and keep on ice.

    Advantages and Features

    Features


    • Assembly and transformation in just under two hours
    • Flexible sequence design (scar-less cloning)
    • No PCR clean-up step required
    • High transformation efficiencies for inserts up to 20 kb
    • Easily adapted for multiple DNA manipulations, including site-directed mutagenesis
    • Includes competent cells

    Properties and Usage

    Materials Required but not Supplied

    DNA Polymerase (for generating PCR products):
    We recommend Q5® High-Fidelity DNA Polymerase (NEB #M0491) or related products, such as Q5 Hot Start High-Fidelity DNA Polymerase (NEB #M0493) or Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494).
    LB (Luria-Bertani) plates with appropriate antibiotic.

    Notes

    1. We highly recommend using our web tool, NEBuilder™ to design PCR primers with overlapping sequences between the adjacent DNA fragments and for their assembly into a cloning vector.

    2. Storage Note:
      The kit is shipped on dry ice. Upon arrival, store kit at -80°C. After first use, store the kit components at indicated temperatures.

    3. Usage notes:

      To ensure the successful assembly and subsequent transformation of assembled DNAs, NEB recommends the following:

      • DNA: PCR product purification is not necessary if the total volume of all PCR products in the Gibson Assembly reaction is 20% or less of the Gibson Assembly reaction volume. Higher volumes of PCR products may reduce the efficiency of Gibson Assembly and transformation due to the elevated carryover amounts of PCR reaction buffer and unused primers present in the PCR product. Column purification of PCR products may increase the efficiency of both Gibson Assembly and transformation by 2–10 fold and is highly recommended when performing assemblies of three or more PCR fragments or assembling longer than 5 kb fragments. Purified DNA for assembly can be dissolved in ddH2O (Milli-Q® water or equivalent is preferable), TE or other dilution buffers.
      • Insert: When directly assembling fragments into a cloning vector, the concentration of assembly fragments should be at least 2–3 times higher than the concentration of vector. For assembly of multiple fragments into a vector, we recommend using equimolar ratio of fragments.
      • Transformation: NEB 5-alpha Competent E. coli (High Efficiency, NEB #C2987) provided with the kit are recommended for use for assembled products of less than 20 kb in size. It is also possible to use other NEB competent E. coli strains, with the exception of BL21, BL21(DE3), Lemo21(DE3) and Nico21(DE3). For example, Shuffle T7 Express Competent E. coli can be used for the expression of a difficult to express protein. When using competent E. coli from a vendor other than NEB, we have seen decreased robustness of transformation with the Gibson Assembly reaction.
      • Electroporation: Electroporation can increase transformation efficiency by several logs. When using the Gibson Assembly Master Mix product for electroporation, it is necessary to dilute the reaction 3-fold and use 1 μl for transformation. Should you require the use of Electrocompetent cells, please use the Electrocompetent Cells Transformation Protocol.

    References

    1. Gibson, D.G. et.al. (2009). Nature Methods. 343-345.
    2. Gibson, D.G. et al. (2010). Nature Methods. 901-903.
    3. Barnes, W.M. (1994). Proc. Natl. Acad. Sci.. 91, 2216-220.

    FAQs

    1. What are the advantages of this method compared to traditional cloning methods?
    2. How large of a DNA fragment can I assemble?
    3. How many fragments of DNA can be assembled in one reaction?
    4. Is this method applicable to the assembly of repetitive sequences?
    5. What are the shortest overlaps that can be used with this assembly method?
    6. What are the longest overlaps that can be used with this method?
    7. Can ≤ 200 bp dsDNA fragments be assembled by this method?
    8. Can ssDNA oligonucleotides be combined and assembled with dsDNA fragments?
    9. Will the reaction work at other temperatures?
    10. Is it necessary to purify PCR products?
    11. Is it necessary to inactivate restriction enzymes after vector digestion?
    12. I would like to produce overlapping dsDNA fragments by PCR. Do I need to use PCR primers that have been purified by PAGE or HPLC?
    13. Can I use a 15-nt overlap that is entirely composed of His-tag repeats (i.e. CACCACCACCACCAC)?
    14. I would like to assemble ssDNA oligonucleotides into dsDNA fragments. Do I need to use oligonucleotides that have been purified by PAGE or HPLC?
    15. Can I PCR-amplify the assembled product?
    16. What should I do if my assembly reaction yields no colonies, a small number of colonies, or clones with the incorrect insert size following transformation into E. coli?
    17. How can I reduce the number of vector-only background colonies?
    18. What type of competent cells are suitable for transformation of DNA constructs created using Gibson Assembly?
    19. Can I use electroporation instead of chemical transformation?
    20. Are there any differences between the Gibson Assembly Master Mix (NEB #E2611) and Gibson Assembly Master Mix included in the Gibson Assembly Cloning Kit (NEB #E5510)?
    21. Are there any differences between the requirements for 2-3 fragment assemblies versus 4–6?
    22. The Gibson Assembly Master Mix control reaction is not giving me any colonies. Why?
    23. When using a polymerase that doesn't contain a 3'-5' exonuclease activity (such as Taq DNA Polymerase) to amplify fragments to be used in a Gibson Assembly reaction, should I be concerned about the potential 3' mismatch generated by the addition of a non-templated nucleotide?
    24. When my Gibson Assembly Cloning Kit arrived, it was stored at -80°C. Will this harm the Gibson Assembly Master Mix?
    25. I would like to use NEBuilder but am concerned about user data privacy. How does NEB handle the information that I enter into NEBuilder?
    26. Can I Use other primer design tools such as SnapGene for Gibson Assembly, to design primers for NEBuilder HiFi DNA Assembly?

    Protocols

    1. Gibson Assembly® Protocol (E5510)
    2. Gibson Assembly® Chemical Transformation Protocol (E5510)
    3. Gibson Assembly® Electrocompetent Cells Transformation Protocol (E5510)

    Manuals

    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

    Selection Tools

    NEB Publications

    • Gutjahr A, Xu SY (2014). Engineering nicking enzymes that preferentially nick 5-methylcytosine-modified DNA Nucleic Acids Res. 42(9), e77. PubMedID: 24609382, DOI: 10.1093/nar/gku192

    Citations

    • 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
    • Lipscomb GL, Schut GJ, Thorgersen MP, Nixon WJ, Kelly RM, Adams MW (2014). Engineering hydrogen gas production from formate in a hyperthermophile by heterologous production of an 18-subunit membrane-bound complex J Biol Chem. 289(5), 2873-9. PubMedID: 24318960, DOI: 10.1074/jbc.M113.530725
    • Guilinger JP, Thompson DB, Liu DR (2014). Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification Nat Biotechnol. 32(6), 577-82. PubMedID: 24770324, DOI: 10.1038/nbt.2909
    • Vandergaast R, Hoover LI, Zheng K, Fredericksen BL (2014). Generation of West Nile virus infectious clones containing amino acid insertions between capsid and capsid anchor Viruses. 6(4), 1637-53. PubMedID: 24721788, DOI: 10.3390/v6041637
    • 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
    • Phelan VV, Moree WJ, Aguilar J, Cornett DS, Koumoutsi A, Noble SM, Pogliano K, Guerrero CA, Dorrestein PC (2014). Impact of a transposon insertion in phzF2 on the specialized metabolite production and interkingdom J Bacteriol. 196(9), 1683-93. PubMedID: 24532776, DOI: 10.1128/JB.01258-13
    • Gai CS, Lu J, Brigham CJ, Bernardi AC, Sinskey AJ (2014). Insights into bacterial CO2 metabolism revealed by the characterization of four carbonic anhydrases in Ralstonia eutropha H16 AMB Express. 4(1), 2. PubMedID: 24410804, DOI: 10.1186/2191-0855-4-2
    • Meinke G, Phelan PJ, Kalekar R, Shin J, Archambault J, Bohm A, Bullock PA (2014). Insights into the initiation of JC virus DNA replication derived from the crystal structure of the T-antigen origin binding domain PLoS Pathog. 10(2), e1003966. PubMedID: 24586168, DOI: 10.1371/journal.ppat.1003966
    • 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
    • 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
    • Chinnici JL, Fu C, Caccamise LM, Arnold JW, Free SJ (2014). Neurospora crassa Female Development Requires the PACC and Other Signal Transduction Pathways, Transcription Factors, Chromatin Remodeling, Cell-To-Cell Fusion, and Autophagy PLoS One. 9(10), e110603. PubMedID: 25333968, DOI: 10.1371/journal.pone.0110603
    • Law SH, Sargent TD (2014). The Serine-Threonine Protein Kinase PAK4 Is Dispensable in Zebrafish: Identification of a Morpholino-Generated Pseudophenotype PLoS One. 9(6), e100268. PubMedID: 24945275, DOI: 10.1371/journal.pone.0100268
    • Royce LA, Boggess E, Fu Y, Liu P, Shanks JV, Dickerson J, Jarboe LR (2014). Transcriptomic analysis of carboxylic acid challenge in Escherichia coli: beyond membrane damage PLoS One. 9(2), e89580. PubMedID: 24586888, DOI: 10.1371/journal.pone.0089580
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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.

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.