Golden gate assembly protocol

For one to three fragments: x1 µl vector DNA

x2 µl insert 1

x3 µl insert 2

x4 µl insert 3

1.0 µl T4 DNA ligase

0.5 µl type IIS enzyme (BsaI, in our case)

1.5 µl 10× ligation buffer

H2O to 15 µl

1. Measure the DNA concentration of insert and vector plasmids nanodrop

2. Add equal amounts of each insert and vector to the reaction mix. Use 20 fmol of each plasmid in a total reaction volume of 15 or 25 µl

3. Calculate volume of vector and insert to use: 20 (fmol) × size (bp) / concentration (ng/µl) / 1520

4. Run Golden Gate Belen method on middle thermocycler