Golden Gate assembly

Golden Gate assembly allows us to create a construct using Type IIs restriction enzymes (REs). These enzymes can generate custom connector sequences, enable scarless assembly, and enhance reaction efficiency through multiple rounds of digestion and ligation.

Protocol for PCR fragments

This protocol is derived from the standard NEB protocol (see citations below).

1. Design the assembly. Online tools such as NEB Golden Gate are available for designing primers, choosing connector sequences, etc.

2. Recipe for Golden Gate reaction (total volume = 10 µL):

Component	Amount
75 ng/plasmid
2:1 mol ratio for fragments
10X T4 DNA ligase buffer	1 µL
T4 DNA ligase (400 U/µL)	1.25 µL (500 U)
Type II RE	0.5 µL (increase to 1 µL if >10 inserts)
H2O	to 10 µL

Note

Make sure to use 500 U T4 DNA ligase. NEB sells 400 and 2,000 U/µL T4 DNA ligase so scale volume accordingly.

3. Use the appropriate assembly protocol

Insert number	Assembly protocol	Estimated time
For 1 insert	37°C, 5 min (cloning) or 37°C, 1 hr (library preparation) -> 60°C, 5 min	10 min - 1 hr
For 2-10 inserts	(37°C, 1 min -> 16°C, 1 min) x 30 -> 60°C, 5 min	~1.5 hrs
For 11-20 inserts	(37°C, 5 min -> 16°C, 5 min) x 30 -> 60°C, 5 min	~5.5 hrs

4. Transform competent cells with 2 µL of the Golden Gate mixture.

5. Store unused Golden Gate assembly products at -20°C

Protocol for pPV plasmids

This protocol is specifically for Golden Gate assembly using the lab’s positioning vector (pPV) cloning scheme and contains general best practices.

1. Design the assembly using the pPV cloning scheme below.

Note

It is okay to combine pPV plasmid fragments with PCR fragments in the same reaction. However, reactions with only plasmid fragments tend to be more efficient.

Important

The Golden Gate reaction will still work if one of the fragments has an internal RE cut site, but it will be less efficient. Be sure that the connector sequence generated by the internal RE cut site does not match any of the pPV connector sequences! If you run into issues with assemblies of this type, try the maximum number of cycles (99x on the thermocycler) and run the reaction overnight. You can also try to DNA clean-up the golden gate reaction and ligate with T4 ligase overnight before transformation.

2. Recipe for Golden Gate reaction (total volume = 10 µL):

Component	Amount
75 ng/plasmid
10X T4 DNA ligase buffer	1 µL
T4 DNA ligase (400 U/µL)	1.25 µL (500 U)
Type II RE	0.5 µL (increase to 1 µL if >10 inserts)
H2O	to 10 µL

Note

Smaller 5 µL (instead of 10 µL) reactions also work, halving the volume of each component. This is best done with multiple, similar reactions by making a master mix containing the ligase buffer, ligase, RE, H20, and any common pPV fragments to avoid pipetting small volumes. However, be sure to use a 10 µL reaction for difficult assemblies (e.g., fragment contains an internal RE cut site).

3. Use the guidelines below to choose an appropriate assembly protocol. In general, longer assemblies (more cycles) are preferred, especially if reactions are run overnight.

Insert	Assembly protocol	Estimated reaction time
<5 plasmid fragments (fast)	(37°C, 1 min -> 16°C, 1 min) x 30 -> 60°C, 5 min	~1.5 hrs
5+ plasmid fragments (fast)	(37°C, 1 min -> 16°C, 1 min) x 60 -> 60°C, 5 min	~3 hrs
5+ plasmid fragments (long), 1 PCR fragment	(37°C, 1 min -> 16°C, 1 min) x 99 -> 60°C, 5 min	~5 hrs
>1 PCR fragments, fragments with internal RE cut site, other difficult assemblies	(37°C, 5 min -> 16°C, 5 min) x 99 -> 60°C, 5 min	~18 hrs

Reactions can be held at 4°C or 16°C overnight.

4. If a fragment contains an internal RE cut site, perform an extra ligation step to improve efficiency.

1. Clean and concentrate the assembly reaction using the DNA Cleanup Kit. Elute in 8.5 µL H20.

2. Add 1 µL 10X T4 DNA ligase buffer and 0.5 µL T4 DNA ligase (400 U/µL), according to the ligation protocol.

3. Let the reaction run at room temperature (or 16°C) at least 1 hr, preferably overnight.

5. Transform competent cells with at least 2 µL of the Golden Gate mixture.

6. Store unused Golden Gate assembly products at -20°C

Reference for pPV connector sequences

Reference for entire Golden Gate workflow

Citations

[1] https://www.neb.com/protocols/2018/06/05/golden-gate-24-fragment-assembly-protocol

[2] https://www.neb.com/-/media/nebus/files/manuals/manuale1601.pdf