Apr 22, 2026
Golden Gate Assembly
- Qinlin Zhang1
- 1Boston University
- Golden Gate Assembly
Protocol Citation: Qinlin Zhang 2026. Golden Gate Assembly. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1rwx7lr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: April 21, 2026
Last Modified: April 22, 2026
Protocol Integer ID: 315421
Keywords: Golden Gate Assembly, golden gate assembly reaction, golden gate assembly golden gate assembly, dna assembly method, dna ligase, type iis restriction enzyme, several dna fragment, restriction enzyme cut, product into competent cell
Abstract
Golden Gate Assembly is a DNA assembly method used to join several DNA fragments in one reaction. It uses a Type IIS restriction enzyme and DNA ligase. The restriction enzyme cuts outside its recognition site, so custom sticky ends can be designed. These sticky ends guide the fragments to join in the correct order. This protocol describes a simple workflow for setting up a Golden Gate Assembly reaction, transforming the product into competent cells, and checking the final construct.
Guidelines
Golden Gate Assembly uses two enzymes in the same tube:
- a Type IIS restriction enzyme such as BsaI
- a DNA ligase such as T4 DNA ligase
The restriction enzyme cuts DNA outside its recognition site and creates designed sticky ends. DNA fragments with matching sticky ends can join together. DNA ligase seals the backbone of the DNA. After correct assembly, the recognition sites are usually removed, so the final product is stable and is not cut again.
Materials
- Plasmid backbone
- DNA insert fragments
- Type IIS restriction enzyme (for example, BsaI)
- T4 DNA ligase
- 10× ligase or assembly buffer
- Nuclease-free water
- Competent E. coli cells
- SOC or LB recovery medium
- LB agar plates with the correct antibiotic
- PCR tubes
- Pipettes and tips
- Ice bucket
- Thermocycler
- Incubator
Before start
Before starting the experiment, check the following:
- Each DNA fragment should have the correct Type IIS recognition sites.
- The overhangs should be designed so that the fragments join only in the desired order.
- Internal recognition sites for the same Type IIS enzyme should be removed if possible.
- DNA concentration should be measured before reaction setup.
Procedure
Prepare DNA Samples
Thaw all reagents on ice.
Mix gently and briefly spin down the tubes.
Measure the concentration of the backbone and insert fragments.
Confirm that the DNA parts match the planned assembly design.
Set Up the Assembly Reaction
Label a PCR tube.
Add backbone DNA, insert fragments, buffer, restriction enzyme, ligase, and water.
Mix gently by pipetting.
Spin down briefly.
Run the Thermocycler
Use a standard Golden Gate cycling program such as:
| Temperature | Duration | |
| 37°C | 2 min | |
| 16°C | 5 min | |
| Repeat these two steps for 25–30 cycles | ||
| 50°C | 5 min | |
| 80°C | 10 min | |
| Hold at 4°C | ||
Transform Competent Cells
Thaw competent cells on ice.
Add 2–5 µL of the assembly reaction to the cells.
Keep on ice for about 20–30 min.
Heat shock if using chemical competent cells.
Add recovery medium.
Incubate the cells for recovery.
Plate the cells on LB agar with the correct antibiotic.
Incubate Plates
Place the plates in an incubator, usually overnight.
Check for colony growth the next day.
Screen Colonies
Pick several colonies.
Use colony PCR, restriction digest, or both to check the construct.
If needed, send positive samples for sequencing.
Expected Results
If the assembly is successful, colonies should appear on the selective plate. Some of these colonies should contain the correct plasmid. Positive colonies should show the expected DNA pattern in colony PCR or digestion analysis. Sequencing can confirm the final result.