NEB® Golden Gate Assembly Mix

Description

The NEB Golden Gate Assembly Mix contains an optimized mix of BsaI and T4 DNA Ligase. Together these enzymes, along with a highly optimized buffer, can direct the assembly of multiple inserts/modules using the Golden Gate approach. Also provided is the pGGA destination plasmid, which provides a backbone for your assembly, and features convenient restriction enzyme sites for subcloning, and has T7/SP6 promoter sequences to enable in vitro transcription.

The efficient and seamless assembly of DNA fragments, commonly referred to as Golden Gate assembly (1,2), has its origins in 1996, when for the first time it was shown that multiple inserts could be assembled into a vector backbone using only the sequential (3) or simultaneous (4) activities of a single type IIS restriction enzyme and T4 DNA ligase.

Type IIS restriction enzymes bind to their recognition sites but cut the DNA downstream from that site at a positional, not sequence-specific, cut site. Thus, a single Type IIS restriction enzyme can be used to generate DNA fragments with unique overhangs. As an example, BsaI has a recognition site of GGTCTC(N1/ N5), where the GGTCTC represents the recognition/binding site, and the N1/ N5 indicates the cut site is one base downstream on the top strand, and five bases downstream on the bottom strand. Assembly of digested fragments proceeds through annealing of complementary four base overhangs on adjacent fragments. The digested fragments and the final assembly no longer contain Type IIS restriction enzyme recognition sites, so no further cutting is possible. The assembly product accumulates with time. 

While particularly useful for multi-fragment assemblies such as Transcription Activator Like Effectors (TALEs)(5) and TALEs fused to a FokI nuclease catalytic domain (TALENs)(6), the Golden Gate method can also be used for cloning of single inserts.  To learn more about the Golden Gate Assembly workflow, watch this video tutorial.

To help select the best DNA assembly method for your needs, please use our Synthetic Biology/DNA Assembly Selection Chart.

Figure 1: Overview: Assembly Protocol of Golden Gate Assembly

Figure 2: Golden Gate Workflow
In its simplest form, Golden Gate Assembly requires a Type IIS recognition site, in this case, BsaI (GGTCTC), added to both ends of a dsDNA fragment. After digestion, these sites are left behind, with each fragment bearing the designed 4-base overhangs that direct the assembly.
Figure 3: NEB Golden Gate Assembly Mix offers improved assembly Figure 3: NEB Golden Gate Assembly Mix offers improved assembly

Assembly reactions were set up using either pre cloned inserts or PCR amplicons directly. Reaction conditions were set up according to manufacturer, and are shown above. Two separate experiments are shown for each reaction type.
Above map shows features of the pGGA plasmid including the cassette that will be excised and replaced by the upon assembly. Unique restriction sites are shown in black. Expanded features section shows location of analysis primers for sequencing or colony PCR, promoter regions, additional restriction sites, the four base BsaI-generated vector backbone overhangs and the sequences of the cassette.

Kit Components

The following reagents are supplied with this product:

Store at (°C)Concentration
NEB® Golden Gate Buffer-2010X
NEB® Golden Gate Assembly Mix-20
pGGA Destination Plasmid-20

Advantages and Features

Features

  • Seamless cloning – no scar remains following assembly
  • Ordered assembly of multiple fragments in a single reaction
  • Can also be used for cloning of single inserts
  • Efficient with regions with high GC content and areas of repeats
  • Compatible with a broad range of fragment sizes (< 100 bp to > 15 kb)
  • Free tool available at GoldenGate.neb.com

Properties and Usage

Materials Required but not Supplied

  • User-defined inserts
  • Competent cells
  • Other materials for transformation

Storage Temperature

-20°C

References

  1. Engler, C. et al (2008). PLoS ONE. 3: e3647.
  2. Engler, C. et al (2009). PLoS ONE. 4: e5553.
  3. Lee, J.H. et al (1996). Genetic Analysis: Biomolecular Engineering. 13; 139-145
  4. Padgett, K.A. and Sorge, J.A. (1996). Gene. 168, 31-35.
  5. Weber, E. et al (2001). PLoS ONE. 6; e19722
  6. Christian, M. et al (2010). Genetics. 186, 757-761.

FAQs

  1. How does NEB Golden Gate Assembly work?
  2. What affects the efficiency of Golden Gate Assembly?
  3. Can PCR amplicons be used directly in assembly reactions without purification?
  4. Why do many of the published Golden Gate Assembly articles feature precloned inserts as opposed to inserts generated by PCR?
  5. Using amplicons directly without precloning seems much easier, but is the assembly efficiency decreased?
  6. Will EDTA interfere with downstream BsaI restriction and ligation?
  7. Why is there a 55°C, 5 min heat step at the end of the assembly reaction?
  8. How can I minimize PCR-generated errors in my amplicon inserts?
  9. Can I use other competent E. coli strain than NEB 10-beta? Can I use subcloning efficiency cells?
  10. Why is Golden Gate also used for single insert cloning?
  11. Can the Golden Gate assembly reactions be scaled down?
  12. I would like to use colony PCR to screen my transformants and sequence my assembly. Are there any recommended sequencing or colony PCR primers?
  13. I’m doing a moderate (4–5 insert) assembly but don’t have access to a thermal cycler; can I use the simpler protocol using the 1 hr 37°C incubation?
  14. For amplicon inserts, why are the calculations suggested as using the overall pGGA destination plasmid length and the overall amplicon lengths? Shouldn’t only the part of pGGA functioning as the vector backbone be used?

Tech Tips

  • Use of the NEB Golden Gate Assembly Tool (GoldenGate.neb.com) is strongly recommended; this tool will check insert sequences for internal BsaI sites and design primers to amplify your inserts for Golden Gate Assembly. The primers will feature 7 bases at the 5´ end flanking the BsaI recognition site, the recognition site itself, plus the 4-base unique overhangs that determine correct annealing and ligation of the inserts. All overhangs will automatically be designed as non-palindromic (to eliminate self insert ligations), unique and in the correct orientations to ensure correct assembly. 

  • Two basic protocol approaches exist for assembly—constant 37°C single temperature incubations, or cycling protocols alternating between 37°C (optimal temperature for endonuclease digestion within a temperature range for ligase stability), and 16°C (optimal temperature for ligation). The assembly protocol suggestions are based on the number of inserts in your assembly reaction, but there is considerable flexibility; match the protocol to your desired efficiency of assembly and scheduling needs. In general: a. Assembly of 1-4 inserts does not require cycling; single 37°C incubations work well. b. Cycling assemblies using 1 min temperature steps work well for multiple inserts. c. Cycling assemblies using 5-10 min temperature steps work well for larger scale assemblies (>10 inserts) and for any assembly for which maximal assembly yields and transformation levels are desired. d. Regardless of the number of inserts, if it is more convenient, any Golden Gate Assembly can be done overnight with 30 cycles of 37°C, 5 min → 16°C, 10 min; i.e., there is no downside for longer protocols being used.

  • Inserts should be screened for the presence of internal BsaI sites. (This is automatically done when using the NEB Golden Gate Assembly Tool, goldengate.neb.com) While inserts with internal BsaI sites can still be used for assembly, the efficiency will be less than if the internal site is removed by silent mutagenesis. If a single internal site is present, omit the final 5 min at 55°C step and screen more colonies to identify your correct assembly.
  • For precloning of inserts, we recommend using the NEB PCR Cloning Kit as the kit's pMiniT 2.0 Vector backbone has no BsaI sites present.
  • While BsaI is blocked by overlapping dcm methylation (methylation at the C5 position of cytosine in the sequences CCAGG or CCTGG), this is usually not an issue for Golden Gate Assembly. Commonly used destination vectors are designed to not contain upstream CC(A or T) bases in front of the BsaI GGTCTC recognition site that would create an overlapping dcm methylation site. 

Protocols

  1. Golden Gate Assembly Protocol for Using NEB Golden Gate Assembly Mix (E1600)
  2. Transformation Protocol for Using NEB Golden Gate Assembly Mix (E1600)
  3. Recommended Screening Protocols for Using NEB Golden Gate Assembly Mix (E1600)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

Selection Charts

Interactive Tools

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.