Golden Gate

Cut-ligation

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 = 1:10 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

[E_coli_Chemical_Transformations | Transform 3–5 μl into E. coli] TOP10 cells.