Golden Gate

Cut-ligation

Following Binder et al 2014.

BsaI cut-ligations

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

• mix 1 μl of each plasmid (each at concentration 100ng/μl)
• 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 0.15 μl of bovine serum albumin (10 mg/ml)

Incubate in a thermocycler using 'GoldenGate' program:

Number of cycles	Temperature	Duration
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.