Gibson cloning

courtesy of the Steckelberg Lab

Making Gibson Master Mix according to Gibson et al. Nature Methods 2010:

5X ISO Reaction Buffer:

• 1.5 mL: 2 M Tris-HCl pH 7.5
• 300 uL: 1 M MgCl2
• 60 uL: Each dNTPs (100 mM Stocks)
• 300 uL: 1 M DTT
• 1.5 g: PEG-8000
• 300 uL: 100 mM NAD Added enough DEPC water to bring volume up to 6 mL (Stored in a 15 mL conical tube in at -20 C)

Gibson Master Mix: (1.2 mL total)

• 320 uL: 5X ISO Buffer
• 0.64 uL: T5 Exonuclease (NEB commercial)
• 20 uL: Phusion (1X homemade stock)
• 160 uL: Taq Ligase (NEB commercial)
• 700 uL: DEPC water

Gibson protocol

• Digest vector with appropriate restriction enzyme(s)
• Clean using Wizard or GeneJet kit
• Put 15 uL Gibson Master Mix in a PCR tube
• Mix in 4 uL digested vector with 1 uL of the gblock (1000 ng dried pellet resuspended in 6 uL DEPC water)*
• Incubate at 50 C for 1 hr
• Transform into DH5a cells

May have to play around with the ratio of vector and inserts to get the right concentrations