Table 3.

PCR cycling parameters for Golden Gate Cloning reaction where BsaI site(s) exist within the insert fragment(s).

37°C 2 min.	25–50X
16°C 5 min.
80°C 10 min.
*16°C 2 h

^*Before the start of this step, 1 μl of fresh DNA ligase needs to be added to the reaction mix.

Destination vector (pGoldenGate-SE7 or pGoldenGate-SE9) is first digested with BsaI, and the non-lacZα fragment of the digestion is gel-purify. The non-lacZα fragment is used in place of the destination vector in the Golden Gate Cloning reaction with the PCR cycling parameter as shown.