Golden Gate Assembly Single Molecule Protocol

Golden Gate Assembly Single Molecule Protocol GGA mixture contains:

1 µL (50 ng) Plasmid

1 µL 10X Promega Ligase Buffer

7 µL dH₂O

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

0.5 µL T4 DNA Ligase to the mixture

10 µL volume

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

• 20 cycles

• 55°C for 10 minutes

• 37°C for 1 minute

• 16°C for 1 minute

• 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.