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.