Gibson assembly is a simple, robust method for assembling multiple DNA fragments without restriction-ligation cloning. Our group routinely uses this method for assembling multiple fragments of DNA into larger constructs, in one step. We generally use Gibson assembly for assemblies of up to ~5 fragments and final construct size of ~20kb; for larger assemblies we usually use yeast homologous recombination.
The method Introduction from Daniel Gibson, et. al., is as follows:
"An isothermal, single-reaction protocol for assembling multiple, overlapping DNA molecules by the concerted actions of a 5’-exonuclease, a DNA polymerase, and a DNA ligase is described. The DNA fragments are first recessed to produce ssDNA overhangs that are specifically annealed, and then they are covalently joined. This assembly protocol can be used to seamlessly construct synthetic and natural genes, genetic pathways, and entire genomes. This method could be a very useful molecular engineering tool."
The original Gibson assembly protocol is here: http://www.nature.com/protocolexchange/protocols/554
The following protocol has minor modifications/optimizations developed that were developed at the Stanford Genome Technology Center (SGTC). They are marked with an asterisk (*) and the reasoning is in italics.
1. 5X Isothermal (ISO) reaction buffer (25% PEG-8000, 500mM Tris-HCl pH 7.5, 50mM MgCl2, 50mM DTT, 1mM each of the 4 dNTPs, and 5mM NAD). This is prepared as described below.
2. T5 Exonuclease (Epicentre)
3. Phusion DNA polymerase (New England Biolabs)
4. Taq DNA Ligase (New England Biolabs)
1. Heat block or thermocycler with PCR tubes.
Make Gibson Master Mixes
1. Prepare ISO Buffer
For 6ml of buffer:
3 mL of 1M Tris-HCl pH 7.5
150 μl of 2M Mgl2
60 μl of 100mM dGTP
60 μl of 100mM dATP
60 μl of 100mM dTTP
60 μl of 100mM dCTP
OR (*alternatively 240ÁL of 100mM dNTPs to individual nucleotides)
300 μl of 1M DTT
300 μl of 100mM NAD
1.5 g of PEG-8000
Add water to 6mL
Aliquot 100 μl into tubes and store at -20 °C.
2. Prepare Master Mix
a. Combine the following on ice:
320 μl of 5X ISO Buffer
20 μl of 2U/μl Phusion polymerase
60 μl of 40U/μl Taq Ligase
6.4 μl of diluted T5 Exonuclease (*Dilute 2 μl of T5 Exonuclease into 18 μl of water to make a 1U/ l mixture. This dilution is recommended as pipetting a small volume of 0.64μl is difficult and can result in more mix to mix variation.)
Add water to 800 μl. (* The orginal protocol was 1.2mL but having more concentrated aliquots allows for more DNA to be added later.)
b. Aliquot 10 μl* into tubes and store at -20°C. (*Alternatively, you can aliquot 5µL and do assembly in a total volume of 10µL once DNA is added)
c. This assembly mixture can be stored at -20 °C for at least one year. The enzymes remain active following at least 10 freeze-thaw cycles. This is ideal for the assembly of DNA molecules with 20-150 bp overlaps. For DNA molecules overlapping by larger than 150 bp, prepare the assembly mixture by using 3.2 μl of 10 U/ μl T5 exo.
1 . Thaw a 10* (*or 5) μl assembly mixture aliquot and keep on ice until ready to be used.
2 . Add 10* (*or 5) μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use 10-100ng of each ~6kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250ng of each 150kb DNA segment).
3 . Incubate at 50°C for 15 to 60 min (60 min is optimal).
3 . If cloning is desired, electroporate 1 μl of the assembly reaction into 30 μl electrocompetent E. coli OR* 2.5µL into Zymo EZ-competent DH5alpha chemically competent E. coli. (*At the SGTC we routinely prepare our own chemically competent cells with this kit).
- Gibson DG , Young L, Chuang R-Y, Venter JC, Hutchison CA, and Smith HO. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 2009 6(5): 343-345. PMID: 19363495