Golden Gate Assembly Protocol for BsmB1

Golden Gate Assembly Protocol GGA mixture contains:

1 µL (50 ng) receiving plasmid

1 µL promoter or other insert at X ng/µL (to determine X ng of insert = (bp insert) (50 ng linearized plasmid-) ÷ (size of entire plasmid in bp)

1 µL 10X NEB Ligase Buffer

6 µL dH₂O

0.5 µL Restriction enzyme (BsmBI-HF, EcoRI-HF, PstI-HF, BsaI-HF, or BbsI)

0.5 µL T4 DNA Ligase from NEB to the mixture

10 µL total volume

Turn on the thermocycler machine. Put tube into machine. Program it for the following cycles:

• 20 cycles of

• 37°C for 1 minute

• 16°C for 1 minute

after 20 cycles:

• 37°C for 15 minutes

• 22°C holding temperature

This DNA ligation is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.

Transformation of GGA For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.