Golden Gate (24 Fragment) Assembly Protocol
- T4 DNA Ligase (NEB #M0202)
- BsaI-HFv2 (NEB #R3733)
- pGGA Destination Plasmid*
- NEB 10-beta Competent E. coli (NEB #C3019)
- NEB 10-beta/Stable Outgrowth Medium (NEB #B9035)
- LB Agar plates with chloramphenicol
* Included in the NEB Golden Gate Assembly Mix (NEB #E1600)
Note: For complex (>10 fragment) assemblies, high efficiencies are achievable with increased ligase and BsaI-HFv2 levels (1000 units T4 DNA Ligase, 30 units BsaI-HFv2), as listed in this protocol. For assemblies involving 10 fragments and less, the standard amounts (500 units T4 DNA Ligase, 15 units BsaI-HFv2) are sufficient. Note the reaction volume of 25 µl is used to allow sufficient volume for precloned insert additions, if needed.
- Set up 25 µl assembly reactions as follows:
REAGENTS ASSEMBLY REACTION NEGATIVE CONTROL (IF DESIRED) pGGA Destination Plasmid*, 75 ng/μl 1 μl (75 ng) 1 μl (75 ng) 24 precloned inserts cloned into pMiniT 2.0, 100 ng/ul each plasmid 0.75 µl (75 ng) each, (18 µl total) - T4 DNA Ligase Buffer (10X) 2.5 μl 2.5 μl T4 DNA Ligase (NEB #M0202), 2000 U/µl 0.5 μl (1000 units) 0.5 μl (1000 units) BsaI-HFv2 (NEB #R3733), 20 U/µl 1.5 μl (30 units) 1.5 μl (30 units) Nuclease-free H2O 1.5 µl 19.5 μl
*or user provided
- Mix gently by pipetting up and down 4 times.
- Briefly microcentrifuge (1 sec.) to bring material to the bottom of tube.
- Transfer to thermocycler and program as follows: (5 min 37°C → 5 min 16°C) x 30 cycles followed by 5 min 60°C. If reactions are done overnight, add a 4°C terminal hold to the protocol, but repeat the final 5 min 60°C step the next day before the transformations.
- For each assembly, thaw a 50 µl tube of NEB 10-beta competent E. coli cells on ice for 5–10 min.
- Add 2 µl of the assembly reaction; gently mix by flicking the tube 4-5 times.
- Incubate on ice for 30 min.
- Heat shock at 42°C for 30 sec.
- Place back on ice for 5 min.
- Add 950 µl of room temperature NEB 10-beta/Stable Outgrowth Medium (NEB #B9035). Incubate at 37°C for 60 min., shaking vigorously (250 rpm) or using a rotation device.
- Warm LB agar plates containing chloramphenicol (for pGGA) at 37°C for 15 min.
- Mix the cells thoroughly by flicking the tube and inverting, then spread 100 µl outgrowth onto each plate.
- Incubate the plates overnight at 37°C, or 24 hrs at 30°C, or 48 hrs at 25°C.