Transformation Protocol for Using NEB Golden Gate Assembly Mix (E1600)

The following protocol is designed for NEB 10-beta Competent E. coli (High Efficiency, NEB #C3019), as this strain is highly efficient for the stable maintenance of large plasmids. For other strains (discussed further in the FAQ section) please refer to the protocol specific to the strain. If using electrocompetent cells, such as NEB 10-beta Electrocompetent E. coli (NEB #C3020), follow the protocol provided with the cells, which can also be found at

For NEB 10-beta Competent E. coli:
  1. Thaw a 50 μl tube of NEB 10-beta Competent E. coli cells on ice for 10 minutes.
  2. Add 2 μl assembly reaction; gently mix by flicking the tube 4–5 times.
  3. Incubate on ice for 30 minutes.
  4. Heat shock at 42°C for 30 seconds.
  5. Place on ice for 5 minutes.
  6. Add 950 μl of room temperature NEB 10-beta/Stable Outgrowth Medium. Incubate at 37°C for 60 minutes, shaking vigorously (250 rpm) or using a rotation device.
Plating Protocol:
  1. Warm LB agar plates containing chloramphenicol (for pGGA) or other appropriate antibiotic at 37°C.
  2. Mix the cells thoroughly by flicking the tube and inverting, then spread 50 μl of a 1:10 dilution (single inserts) or 50–100 μl (multiple inserts) of the 1 ml outgrowth onto each plate.
  3. Incubate the plate overnight at 37°C, or if desired, 24–36 hours at 30°C or 48 hours at 25°C.
  1. KuceraBecky

    Restriction Enzymes in Golden Gate Assembly

    Type IIS restriction enzymes have both recognition and binding sites, but cut downstream of the recognition site, creating 4-base overhangs ideal for re-assembly. View a list of TypeIIS enzymes.