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.
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
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.
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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.
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
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