Home Protocols Golden Gate (24 Fragment) Assembly Protocol

Golden Gate (24 Fragment) Assembly Protocol

Materials

  • 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.

 

Assembly Reactions

  1. 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

  2. Mix gently by pipetting up and down 4 times.

  3. Briefly microcentrifuge (1 sec.) to bring material to the bottom of tube.

  4. 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. 

 

Transformation

  1. For each assembly, thaw a 50 µl tube of NEB 10-beta competent E. coli cells on ice for 5–10 min.

  2. Add 2 µl of the assembly reaction; gently mix by flicking the tube 4-5 times.

  3. Incubate on ice for 30 min.

  4. Heat shock at 42°C for 30 sec.

  5. Place back on ice for 5 min.

  6. 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.

 

Plating

  1. Warm LB agar plates containing chloramphenicol (for pGGA) at 37°C for 15 min.

  2. Mix the cells thoroughly by flicking the tube and inverting, then spread 100 µl outgrowth onto each plate.

  3. Incubate the plates overnight at 37°C, or 24 hrs at 30°C, or 48 hrs at 25°C.