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  • Gibson Assembly® Master Mix

    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 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).

    Overview of the Gibson Assembly 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. 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 Master Mix 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 using agarose gel electrophoresis, a Nanodrop™ instrument or other method
    • Add DNAs 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 E. coli or use directly in other applications

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuilder® Positive Control-202X
    Gibson Assembly® Master Mix-20*2X

    * Reagents' Storage Notes

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

    Advantages and Features

    Features


    • Increased number of successful assembly products, particularly for longer or greater number of fragments
    • Flexible sequence design (scarless cloning)
    • No clean-up step required
    • Complex assembly achieved in 1 hour
    • DNA can be used immediately for transformation, or as template for PCR or RCA
    • Easily adapted for multiple DNA manipulations, including site directed mutagenesis, insertions and deletions

    Properties and Usage

    Materials Required but not Supplied

    DNA Polymerases (for generating PCR products):
    We recommend Q5® High-Fidelity DNA Polymerase (NEB #M0491) or related products, such as Q5 Hot Start Flex DNA Polymerase (NEB #M0493), Q5 Hot Start Flex 2X Master Mix (NEB #M0494).

    LB (Luria-Bertani) plates with appropriate antibiotic.

    SOC Outgrowth Medium (NEB #B9020).

    Competent Cells:
    We recommend NEB 5-alpha Competent E. coli (High Efficiency, NEB #C2987). For assembled products greater than 10 kb, NEB recommends using NEB 10-beta Competent E. coli (High Efficiency, NEB #C3019) or NEB 10-beta Electrocompetent E. coli (NEB #C3020).

    Storage Temperature

    -20°C

    Storage Notes

    • Store at -20°C. Thaw, vortex thoroughly before use and keep on ice.

    Notes

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

    2. Usage notes:

      To ensure the successful assembly and subsequent transformation of assembled DNAs, NEB recommends the following:
      • Cells: Transformation efficiency of competent cells can vary by several logs. Perceived assembly efficiency directly correlates to the competence of the cells used for transformation.
      • 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.
      • 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 2–3 times higher than the concentration of vector. For assembly of 3 or more fragments, we recommend using equilmolar ratio of fragments.
      • Biology: Some DNA structures, including inverted and tandem repeats, are selected against by E. coli. Some recombinant proteins are not well tolerated by E. coli and can result in poor transformation or small colonies.

    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, 2216-2220.

    FAQs

    1. What are the advantages of this method compared to traditional cloning methods?
    2. How large 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. Can longer or shorter incubation times be used?
    10. Will the reaction work at other temperatures?
    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. I would like to assemble ssDNA oligonucleotides into dsDNA fragments. Do I need to use oligonucleotides that have been purified by PAGE or HPLC?
    14. Can I use a 15-nt overlap that is entirely composed of His-tag repeats (i.e. CACCACCACCACCAC)?
    15. Can you 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. Is storing Gibson Assembly Master Mix at -80°C harmful?
    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® Master Mix – Assembly (E2611)
    2. Gibson Assembly® Chemical Transformation Protocol (E2611)
    3. Gibson Assembly® Electrocompetent Cells Transformation Protocol (E2611)

    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].

    Application Notes

    NEB Publications

    • Gutjahr A, Xu SY (2014) 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.

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Functional Test (Gibson Assembly):
      The Gibson Assembly Master Mix is incubated with linearized pUC19 and five 400 bp fragments containing 40 bp overlaps. The mixture is transformed into E. coli and the assembled five fragment insert is verified by colony PCR and agarose gel electrophoresis resulting in the expected 2000 bp product.

    Safety Data Sheet

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