Method Details
Reaction Setup
Component | Volume | Notes |
|---|---|---|
| BsaI/Esp3I/BbsI | 0.5 µL | 0.5–0.75µL range. More does not help. |
| T4 DNA Ligase | 0.5 µL | 0.5–1 µL range, 2000 CEU/µL. cligase ∝ misligation. |
| 10× T4 Ligase Buffer | 1.5 µL | Titurate/vortex to dissolve solids. |
| 10× BSA | 1.5 µL | Enables full BsaI activity at 37°C |
| DNAs | 25 fmol each 0.5 µL 50 nM | 10–40 fmol equimolar. 2–5-fold less vector to reduce background. |
| Deionized Water | up to 15 µL | 10–20 µL range. |
Thermocycling Conditions
BsaI Golden Gate, Long, ≥6 parts: 142.5 min / 2:23 | Basic*: 52.5–75 min | ||||||
| Step | Temp | Time | Temp | Time | |||
|---|---|---|---|---|---|---|---|
| Initial Digestion (opt.) | 37°C | 10 min | 37°C | 15 min | |||
Repeat 25× / 15× | Digestion | 37°C | 1.5 min | Repeat 5–10× | 37°C | 1.5 min | |
| Annealing & Ligation | 16°C | 3 min | 16°C | 3 min | |||
| Digestion & Ligase Inact. | 50°C | 10 min | 50°C | 5 min | |||
| Inactivation | 80°C | 10 min | 80°C | 10 min | |||
| Storage | 12°C | ∞ | 12°C | ∞ | |||
Esp3I Golden Gate, Long, ≥6 parts: 142.5 min / 2:23 | Basic*: 52.5–75 min | ||||||
| Step | Temp | Time | Temp | Time | |||
|---|---|---|---|---|---|---|---|
| Initial Digestion (opt.) | 37°C | 10 min | 37°C | 15 min | |||
Repeat 25× / 15× | Digestion | 37°C | 1.5 min | Repeat 5–10× | 37°C | 1.5 min | |
| Annealing & Ligation | 16°C | 3 min | 16°C | 3 min | |||
| Digestion | 37°C | 5 min | 37°C | 5 min | |||
| Digestion & Ligase Inact. | 50°C | 5 min | 50°C | 5 min | |||
| Inactivation | 80°C | 10 min | 80°C | 10 min | |||
| Storage | 12°C | ∞ | 12°C | ∞ | |||
Use the long protocol whenever you have time or trouble, as it is most efficient.
* Basic protocol is said to be sufficient for basic part assemblies, which can also be run isothermal, 37°C 1 hr. Reactions containing only 2–3 linear fragments are especially fine with the basic protocol.
If one or more targeted restriction sites needs to be preserved in the product, it is best to omit final restriction steps and end the thermocycling in ligation-permissive conditions to maximize ligation at the retained restriction site. Lack of religation greatly reduces efficiency of this kind of Golden Gate assembly. Usage of a different type IIs enzyme could be possible instead.
Golden Gate End-On-Ligation | |||
| Step | Temp | Time | |
|---|---|---|---|
| Initial Digestion (opt.) | 37°C | 10 min | |
Repeat 25× | Digestion | 37°C | 1.5 min |
| Annealing & Ligation | 16°C | 3 min | |
| Storage | 16°C | ∞ | |
Golden Gate Assembly Research
CIDAR MoClo Protocol
Densmore Lab CIDAR MoClo protocol, from supplement:
| |||||||||||||||
| Standard, ≤5 part | Troubleshooting, ≥7 part | Rapid, for basic part construction | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0 | opt. 37°C | 0/20 min | opt. 37°C | 0/20 min | 37°C | 20 min | |||||||||
| 1 | 37°C | 1.5 min | 37°C | 1.5 min | 37°C | 1.5 min | |||||||||
| 2 | 16°C | 3 min | 16°C | 3 min | 16°C | 3 min | |||||||||
| 3 | Cycle 1–2 | 15× | Cycle 1–2 | 25× | Cycle 1–2 | 5–10× | |||||||||
| 4 | 50°C | 5 min | 50°C | 5 min | 50°C | 5 min | |||||||||
| 5 | 80°C | 10 min | 80°C | 10 min | 80°C | 10 min | |||||||||
| Total | 82.5 min | 127.5 min | 57.5–80 min | ||||||||||||
| Transform 2–5 µL | |||||||||||||||
"Three protocols have been developed to optimize reaction time and cloning efficiency. The Standard Protocol provides >80% efficiency (>80% of all clones are correct with more than 200 colonies per plate in an 82.5 minute reaction time. It is ideal for simple 4-part + vector assemblies. The Troubleshooting protocol is used for more than 6 part + vector reactions and in Multiplexed MoClo to provide a larger number of correctly assembled clones. The Rapid protocol was designed for quickly assembling basic parts from PCR product or annealed oligos where only one part is being ligated to a vector. The longer initial digestion time could also be adopted in the Standard and Troubleshooting protocols to increase efficiency if needed."[1]
DNA Ligase
- T4 DNA ligase can ligate blunt ends (undesirable for Golden Gate).
- T7 DNA ligase cannot, only efficiently ligating ≥2 bp annealed ends under normal conditions.
- T4 ligase has optimal activity 16–20°C, permissive to cohesive end annealing at 14–16°C.
- T7 ligase has optimal activity at 25°C, much less permissive to cohesive end annealing. A few degrees makes a big difference for 4 nt, 25% G/C.
Buffer-protocol compatibility
- T4 ligase buffer is compatible with heat inactivation and electrotransformation.
- T7 ligase buffer contains PEG3000; 65°C heat inactivation dramatically reduces transformation efficiency and the reaction can neither be electroporated.
- T7 ligase only has 10% activity in T4 ligase buffer.
- High specificity for correctly base-paired nicks if T7 ligase used with T4 ligase buffer instead of supplied T7 ligase buffer.[NEB]
Buffer strengths
- NEB and Promega T4 ligase buffers are 10×, allowing more DNA volume in a small reaction.
- NEB T7 ligase buffer is 2×, allowing 4 µL DNA volume in a typical 10 µL rxn with 1 µL total enzyme.
Inactivation
- T4 and T7 ligases are heat-inactivated at 65°C in 10 min.
Type IIS Restriction Endonucleases
Here are all commercial ≥3 bp overhang-creating type IIs restriction endonucleases, and analysis of their properties for Golden Gate assembly.
BsaI
- Instructed to be used with BSA (bovine serum albumin) in old buffer system, indicating that it is ≈essential at the recommended reaction temperature, 37°C. The new buffer system adds BSA to all standard restriction enzyme buffers for simplification, as it almost never reduces activity of enzyme that don't benefit from it.
- NEB writes that BsaI digest does not require BSA for 100% activity at 50°C, but does for activity at the lower 37°C.
- Neither T4 nor T7 ligase buffers of Promega or NEB contain BSA. Though the PET in T7 ligase buffer may function similarly.
- J5 Golden Gate protocol says that BsaI only has 10% activity at 37°C without BSA (bovine serum albumin), and thus requires adding BSA to Golden Gate reactions, as their protocol is performs digestion at 37°C.
- The CIDAR protocol seems to have overlooked BSA or discounted its utility in their optimization.
- The Dueber Lab does not add BSA, and performs the digestion step at 45°C, closer to 50°C optimum but lower so as to less hurt the ligase. But Shyam did otherwise after research.
- BsaI requires a full unit to digest a microgram of substrate in one hour or sixteen hours, meaning that it's active for only one hour.[NEB)
- Impaired by Dcm methylation at CCWGGTCTC sites. NEB
- NEB's BsaI-HF was found to have lower efficiency cutting at sites with an A/T spacer and generally performs worse in Golden Gate assembly.
BsmBI
- NEB recommends reaction at 55°C, but such may harm the ligase. CIDAR MoClo doesn't use BsmBI, so doesn't make a recommendation.
- Dueber Lab performs the digestion step at 45°C, 10° off optimum to less hurt the ligase, but it seems to work well.
- Not instructed to be used with BSA in the old buffer system; therefore BSA is not required.
- BsmBI requires only 0.5 U to completely digest a microgram of substrate in sixteen hours, but cannot with 0.25 U, meaning that it's active between two and four hours.[NEB]
BbsI BpiI
- Has ≤25% activity at temperatures ≤25°C, according to NEB. Thus, some activity during the ligation step.
- Store at -80°C, not -20°. NEB tested full activity after ten freeze-thaws. Storing and using aliquots is safest.
- BbsI-HF is improved to allow storage at -20°C.
- Not instructed to be used with BSA in the old buffer system; therefore BSA is not required.
- BpiI is an isoschizomer of BbsI
Esp3I
- Mesophilic isoschizomer of BsmBI
- Temperature optimum is 37°C.
- Functions much better for Golden Gate assembly than problematic NEB BsmBI lots.
- 2.5% PEG-3350 in the reaction can improve Golden Gate efficiency 2-fold for a ten part assembly. NEB warns not to heat-inactivate reactions containing PEG, but this improvement was detected with the typical 80°C, 10 min heat-inactivation.
Eco31I
- Mesophilic isoschizomer of BsaI.
- Untested for superiority over BsaI.
BtgZI
- Cuts 10 bp away from recognition, used optionally in GoldenBraid to cut over a BsaI site to release the same overhang. This could allow, say, a BsmBI-reactive multigene acceptor vector to be used in a BsaI cassette assembly, or vice-versa.[GoldenBraid]
- 100% activity at 60°C, 75% activity at 37°C.
- Ineffective longer than 1 hr in rxn, like BsaI.
AarI
- 7 bp recognition site
- Requires unique buffer containing an oligonucleotide for optimal digestion, indicating its need for two sites for cleavage.
- 39-fold as expensive as BsaI by activity.
BfuAI BspMI BveI
- Require two sites on the DNA to cleave.
- BfuAI cleaves plasmid DNAs more efficiently than BspMI.
- BfuAI requires 50°C, with 50% activity at 37°C.
- Oligonucleotide provided with BveI to add to reaction and assist cleavage, probably releasing unwanted overhangs.
SapI BspQI LguI
- 7 bp recognition site
- 3 bp overhang
- SapI and BspQI are ineffective longer than 1 hr in rxn, like BsaI.
EarI Eam1104I
- 3 bp overhang
- Active >8 hr.