Gibson Assembly

Gibson assembly is a DNA assembly method which allows to join multiple DNA fragments together in a single, isothermal reaction. This method was invented by Daniel Gibson in 2009.

A single-tube Isothermal Assembly reaction features three different enzymatic activities that perform in the same 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.

Isothermal Assembly works by combining a cocktail of exouclease, polymerase, an ligase to fuse dsDNA fragments with sufficiently (20-120 bp) homologous ends. It leaves no "scar" behind, i.e. you can expect your product to contain the EXACT over lap sequence. The reaction may work with shorter ends (e.g. 15 bp), so long as the annealing temperature is higher than 50°C.

To perform isothermal assembly:

1. PCR up your fragments of choice, and gel purify. Also gel purify the cut vector

2. Not exceeding a total volume of 5 µL, in a PCR tube, combine fragments at equal molecular ration [e.g. amount fragment 1 = 100 ng * (fragment size 1 / fragment size 2); amount fragment 2 = 100 ng* (fragment size 2 / fragment size 1)... etc.]. If required, bring to 5 µL with ddH2O. We recommend using approximately 100 ng of plasmid backbone.

3. Add the combined fragments (5 µL) to one Isothermal Assembly reaction aliquot (15 µL) and mix by pipetting (20 µL total).

4. Incubate samples in a thermocycler at 50°C for 60 minutes

5. (optional for chem. transformation)Purify with Qiagen PCR purification (MinElute) kit. Elute in 20 µL of ddH2O. [or simply dialyse]

6. The end result is a circular, double-stranded, fully-sealed DNA molecule that can be used to transform into Competent E. coli by using 10 µL of assembly mix

Making Isothermal Assembly Aliquots

5x isothermal assembly reaction buffer (assemble on ice):

From the paper: 	Actually added: 
3 mL 1M Tris-HCl pH 7.5 	3 mL 1M Tris-HCl pH 7.5 
150 uL 2M MgCl2 	300 uL 1M MgCl2 
60 uL 100 mM dGTP 	600 uL 10 mM each dNTP 
60 uL 100 mM dCTP 	  
60 uL 100 mM dTTP 	  
60 uL 100 mM dATP 	  
300 uL 1M DTT 	300 uL 1M DTT 
1.5 g PEG-8000 	1.5 g PEG-8000 
300 uL 100 mM NAD 	20 mg NAD 
ddH2O to 6 mL 	ddH2O to 6 mL 

• Prepare 320 uL aliquots (18) and freeze all but one. * Label these “5X isotherm buffer”

To the one remaining (320 uL), add: 1.2 uL T5 Exonuclease 20 uL Phusion polymerase (NOT HOTSTART) 160 uL Taq ligase 700 uL ddH2O Prepare 15 uL aliquots (~80) on ice in PCR tubes and store at -20C. These should be good for up to a year.

Shopping List Buffer Item Vendor Cat. No. 1M Tris-HCl pH 7.5 Systembio kitchen none Magnesium Chloride, 1.00 +/- 0.01M Solution Affymetrix / USB 78641 10 x 1 ML Nicotinamide adenine dinucleotide (NAD) Applichem A1124,0005 DTT, molecular biology grade FERMENTAS R0861 Polyethylene Glycol 8000, Powder USB / Affymetrix 19966 dNTP Mix, 10mM each Fermentas R0192 1 ml Enzymes: T5 Exonuclease EPICENTRE T5E4111K Taq DNA Ligase NEB M0208L Phusion™ High-Fidelity DNA Polymerase NEB F-530S

References: Gibson et al (2009) Nature Methods 6(5):343-345.