This page contains the recipe and reaction condition for assembling multiple overlapping fragments.

Read this article for an overview of Gibson Assembly and primer design.

5X ISO buffer. This recipe makes 6ml.

• 3 ml of 1 M Tris-HCl pH 7.5
• 150 μl of 2 M MgCl₂
• 60 μl of 100 mM dGTP
• 60 μl of 100 mM dATP
• 60 μl of 100 mM dTTP
• 60 μl of 100 mM dCTP
• 300 μl of 1 M DTT
• 1.5 g PEG-8000
• 300 μl of 100 mM NAD
• Add water to 6 ml
• Aliquot 100 μl and store at -20 °C

Gibson Assembly Enzyme Mix. This recipe makes 1.2ml

• 320 μl 5X ISO buffer
• 0.64 μl of 10 U/μl T5 exonuclease
• 20 μl of 2 U/μl Phusion polymerase
• 160 μl of 40 U/μl Taq ligase
• Add water to 1.2 ml
• Aliquot 15 μl and store at -20 °C.

Protocol:

1. Thaw a 15 μl aliquot of Gibson Assembly Enzyme Mix on ice.

2. Add 5 μl of DNA (equimolar amounts of each fragment to a total of 50-200 ng)

3. Mix gently.

5. Incubate at 50 °C for 15 to 60 min.

6. Transform 1 – 5 μL.