Gibson Assembly® Master Mix
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Gibson Assembly allows for successful assembly of multiple DNA fragments, regardless of fragment length or end compatibility.
- 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
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Product Information
Gibson Assembly 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 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 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
- This product is related to the following categories:
- DNA Assembly, Cloning and Mutagenesis Kits
- This product can be used in the following applications:
- Gibson Assembly
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Protocols, Manuals & Usage
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Tools & Resources
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FAQs & Troubleshooting
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Citations & Technical Literature
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Additional Citations
- 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
- 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
- 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
- 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
- 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
- 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
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