Protocols · Benchling

Materials

NEBridge^® Golden Gate Enzyme Mix (BsaI-HFv2)
T4 DNA Ligase Reaction Buffer
pGGAselect DNA, or other destination vector

Inserts
Nuclease-free water

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.

10× BSA + PEG enhancer (opt.)	1 µL or 1×	10× = 1 mg/mL BSA + 10% PEG-3350 Golden Gate enhancer.
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.
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.
DNA parts	25 fmol each, 0.5 µL 50 nM	10–40 fmol range, equimolar. 2-fold less vector to reduce vector religation background.
PaqCI activator, 20 µM	0.25 µL	Use only in PaqCI/AarI assemblies.
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).
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.

BsaI/PaqCI Cycling Golden Gate Long, ≥6 parts: 2:23; Short, ≤5 parts: 1:38	BsaI/PaqCI Cycling Golden Gate Long, ≥6 parts: 2:23; Short, ≤5 parts: 1:38	BsaI/PaqCI Cycling Golden Gate Long, ≥6 parts: 2:23; Short, ≤5 parts: 1:38	BsaI/PaqCI Cycling Golden Gate Long, ≥6 parts: 2:23; Short, ≤5 parts: 1:38	Basic, 2–3 parts: 0:52–1:15	Basic, 2–3 parts: 0:52–1:15	Basic, 2–3 parts: 0:52–1:15
	Step	Temp	Time		Temp	Time
Lid: 75°	Initial Digestion (opt.)	37°C	10–20 min		37°C	20 min
Repeat (25× for Long / 15× for Short)	Digestion	37°C	1.5 min	Repeat (5–10×)	37°C	1.5 min
Repeat (25× for Long / 15× for Short)	Annealing & Ligation	16°C	3 min	Repeat (5–10×)	16°C	3 min
	Digestion & Ligase Inact.	50°C	10 min		50°C	5 min
	Inactivation	65°C	10 min		80°C	5 min