Optimized Golden Gate Assembly
Introduction
Materials
- NEBridge® Golden Gate Enzyme Mix (BsaI-HFv2)
- T4 DNA Ligase Reaction Buffer
- pGGAselect DNA, or other destination vector
- Inserts
- Nuclease-free water
Procedure
Golden Gate Assembly
- Thaw Golden Gate Assembly reagents, inserts, and backbone on ice.
- Label a PCR tube and keep it on ice
- Briefly vortex 10× T4 Ligase Buffer
- Calculate the required volumes of the backbone and inserts using this Google Sheet calculator, aiming for 25pmol of inserts and 12.5pmol of backbone.
- If calculated volumes are large, reduce the molar concentration of the inserts to 15pmol, and subsequently reduce the molar concentration of the backbone to 7.5pmol.
- Calculate the required volumes of the 10× T4 Ligase Buffer, master mix, and deionized water.
A A | B B | C C | |
1 1 | 10× BSA + PEG enhancer (opt.) | 1 µL or 1× | 10× = 1 mg/mL BSA + 10% PEG-3350 Golden Gate enhancer. |
2 2 | 10× T4 Ligase Buffer | 1 µL or 1× | Triturate/vortex to dissolve DTT precipitates. Limit freeze-thaws by keeping aliquot at 4° for ≤1 yr. |
3 3 | Deionized Water | up to 10 µL | 7–20 µL range. Enzymes ≤10% rxn vol. Aim for DNA being <½ the reaction. Rxns master mixes can be split for even smaller volumes. |
4 4 | DNA parts | 25 fmol each, 0.5 µL 50 nM | 10–40 fmol range, equimolar. 2-fold less vector to reduce vector religation background. |
5 5 | PaqCI activator, 20 µM | 0.25 µL | Use only in PaqCI/AarI assemblies. |
6 6 | T4 DNA Ligase (400 CEU/µL, not high-conc) | 0.2 µL | 0.1–0.5 µL range, 2000 CEU/µL. Hi-T4also works. Use ~10 CEU per DNA part = 0.025 µL ligase. cligase ∝ misligation (1). |
7 7 | Type IIs endonuclease BsaI, PaqCI, Esp3I/BsmBI, BbsI, SapI | 0.5 µL | 0.2–0.75 µL range. More does not help (1). Use 1 µL Esp3I/BsmBI for complex assemblies. Use ~1 U per DNA part = 0.05 µL BsaI, 0.1 µL PaqCI/Esp3I. |
- Assemble all reaction components on ice/cold block in thermocycler "PCR" tubes.
- Enzymes must be added after at least buffer and water are mixed: combine water, buffer, enhancers and mix; then add endonuclease and ligase and mix.
- As enzymes are in viscous 50% glycerol, too much enzyme will be aspirated if tip is well below the liquid surface.
- Make master mixes when possible, as it reduces pipetting steps, reduces errors from pipetting small volumes, and maximizes component precision across reactions. Combine all common components for n reactions, and aliquot volumes reduced by the volume of the variable components, which are generally one or more DNA parts. Wetlab Calculator.
- Watch that all components enter and exit the pipette tip to ensure no component fails to be transferred. Even one missing 0.5µL part will ruin the reaction.
- Make 2–5% extra master mix to account for pipetting error.
- After adding last component, mix reaction by pipetting or by flicking and centrifuging tubes to recollect liquid at bottom.
- Thermocycle/incubate according to reaction complexity and time constraints.
A A | B B | C C | D D | E E | F F | G G | H H | |
1 1 | BsaI/PaqCI Cycling Golden Gate
Long, ≥6 parts: 2:23; Short, ≤5 parts: 1:38 |
Basic, 2–3 parts: 0:52–1:15 | ||||||
2 2 | Step | Temp | Time | Temp | Time | |||
3 3 | Lid: 75° | Initial Digestion (opt.) | 37°C | 10–20 min | 37°C | 20 min | ||
4 4 | Repeat (25× for Long / 15× for Short) | Digestion | 37°C | 1.5 min | Repeat (5–10×) | 37°C | 1.5 min | |
5 5 | Repeat (25× for Long / 15× for Short) | Annealing & Ligation | 16°C | 3 min | Repeat (5–10×) | 16°C | 3 min | |
6 6 | Digestion & Ligase Inact. | 50°C | 10 min | 50°C | 5 min | |||
7 7 | Inactivation | 65°C | 10 min | 80°C | 5 min | |||
Golden Gate Assembly is a molecular cloning method that allows for the precise and efficient assembly of multiple DNA fragments in a single reaction. This technique utilizes Type IIS restriction enzymes, which cut DNA at sites outside of their recognition sequences, creating overhangs that can be seamlessly ligated together. By designing the overhangs to be unique and complementary, multiple fragments can be assembled in a specific order in one step. This method is highly efficient for assembling multiple genes or DNA parts into a plasmid backbone, making it a popular choice for synthetic biology and genetic engineering applications.