Golden Gate

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Following Binder et al 2014. and Kakui et al 2015.

Dilute each module element (including linear modules such as the GOI) in 20 fmol/μl:

  • Measure the DNA concentration with the NanoDrop
  • Obtain the length of the plasmid or fragment
  • Calculate the dilution to obtain 13.3 ng/μl per 1-kilobase DNA (for a plasmid: insert length + 3.5kb)
  • Make the dilutions

BsaI cut-ligations

For a total of 15 μl reaction, while working on ice:

  • mix 1 μl of each plasmid
  • add 0.5 μl BsaI (5 units)
  • add 0.75 μl T4 ligase (NEB)
  • add 1.5 μl ligase buffer (avoid freeze-thaw cycles to protect the ATP)
  • add 0.75 μl of bovine serum albumin (2 mg/ml = 1:5 dilution from NEB)

Incubate in a thermocycler using 'GoldenGate' program:

Number of cycles Temperature Duration
1X 37°C 10 min
40X 37°C 2 min
16°C 5 min
1X 37°C 5 min
50°C 5 min
80°C 5 min
7°C Inf

Transform 3–5 μl into E. coli TOP10 cells.