Protocol Code: A1

Gibson Assembly

5x isothermal buffer

• 0.75g - 25% PEG-8000
• 1500 µL - 500 mM Tris-HCl pH 7.5
• 75 µL - 50mM MgCl₂
• 150 µL - 50mM DTT
• 30 µL - 1mM dATP
• 30 µL - 1mM dTTP
• 30 µL - 1mM dCTP
• 30 µL - 1mM dGTP
• 300 µL - 5mM NAD
• Nuclease-free water to fill to 3000µL

Total - 3000 µL

Gibson Master Mix

• 50 µL - Taq ligase (40u/µL)
• 100 µL - 5x isothermal buffer
• 2 µL - T5 exonuclease (1u/µL)
• 6.25 µL - Phusion polymerase (2u/µL)
• 216.75 µL - Nuclease-free water

Total - 375 µL