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Eleven Tips For Optimizing Your
Golden Gate Assembly Reactions
Looking to assemble multiple DNA fragments in a single reaction? Here are some tips
to keep in mind when planning your Golden Gate Assembly experiment.

Australia & New Zealand
New England Biolabs (Australia) PTY
Telephone: +61 (1800) 934218
info.au@neb.com

1 Check your sequences

4 Choose the right buffer

8 Avoid PCR-induced errors

Always check your assembly sequences for internalsites before choosing which Type IIS restriction
endonuclease to use for your assembly. While
single insert Golden Gate Assembly has such highefficiencies of assembly that the desired product isobtainable regardless of the presence of an internalsite, this is not true for assemblies with multiple
inserts. Options include choosing a different
Type IIS restriction enzyme to direct your assembly,
or eliminating internal sites through domestication.
Our tutorial video on Golden Gate AssemblyDomestication provides a full description of themany options available for internal site issues. Notethe use of a Type IIS restriction enzyme with a 7base recognition site, such as PaqCI, is less likely tohave internal sites present in any given sequence.

T4 DNA Ligase Buffer works best for Golden Gate
Assembly with BsaI-HFv2, BsmBI-v2 and PaqCI.
However, alternate buffers would be NEBuffer r1.1
for Bsa-HFv2, NEBuffer r2.1 for BsmBI-v2, orrCutSmart™ Buffer for PaqCI, if these buffers aresupplemented with 1 mM ATP and 5–10 mM
DTT. NEB also offers NEBridge Ligase Master Mix
that has been optimized for Golden Gate Assemblywith our Type IIS restriction enzymes
for Golden Gate.

Do not over-cycle and use a proofreading highfidelity DNA polymerase, such as Q5® DNA HighFidelity Polymerase.

CanadaNew England Biolabs, Ltd.
Toll Free: 1-800-387-1095
info.ca@neb.com

2

Orient your primers

When designing PCR primers to introduce TypeIIS restriction enzyme sites, either for ampliconinsert assembly or as an intermediate for precloning the insert, remember that the recognition
sites should always face inwards towards yourDNA to be assembled. Consult the Golden Gate
Assembly Kit manuals or assembly videos forfurther information regarding the placement andorientation of the sites.

3 Choose the right plasmid
Consider switching to the versatile pGGAselectDestination Plasmid for your Golden Gate
Assembly. This versatile new destination construct
is included in all Golden Gate Assembly kits andcan be used for BsaI-HFv2, BsmBI-v2 or BbsI
directed assemblies. It also features T7 and SP6
promoter sequences flanking the assembly site,and has no internal BsaI, BsmBI or BbsI sites.
The pGGAselect plasmid can also be transformed
into any E. coli strain compatible with pUC19 forproducing your own plasmid preparation if sodesired.

ncrease your complex assembly
5 Iefficiency
by increasing the Golden
Gate cycling levels
T4 DNA Ligase, BsaI-HFv2, BsmBI-v2 and PaqCI
are very stable and continue to be active duringextended cycling protocols; an easy way to increase
assembly efficiencies without sacrificing fidelity
is to increase the total cycles from 30 to 45–65,even when using long (5-minute) segments for thetemperature steps.

ake sure your plasmid prep is
6 MRNA-free
For pre-cloned inserts/modules, make sure
your plasmid prep is free of RNA to avoid anoverestimation of your plasmid concentrations.

7 Avoid primer dimers
For amplicon inserts/modules, make sure yourPCR amplicon is a specific product and contains
no primer dimers. Primer dimers, with Type IIS
restriction endonuclease sites (introduced in theprimers used for the PCR reactions), would be
active in the assembly reaction and result inmis-assemblies.

ecrease insert amount for
9 Dcomplex
assemblies

China
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France
New England Biolabs France SASTelephone: 0800 100 632
info.fr@neb.com
Germany & Austria
New England Biolabs GmbHFree Call: 0800/246 5227 (Germany)
Free Call: 00800/246 52277 (Austria)
info.de@neb.com
Japan
New England Biolabs Japan, Inc.
Telephone: +81 (0)3 4545 1422
cs.jp@neb.com

Now includes NEBridge® Ligase Master Mix

Golden Gate Assembly
50+ FRAGMENT ASSEMBLY NOW ACHIEVABLE
WITH HIGH EFFICIENCY AND ACCURACY

Singapore
New England Biolabs, PTE. Ltd.
Telephone: +65 638 59623
sales.sg@neb.com
United Kingdom
New England Biolabs (U.K.) Limited
Call Free: 0800 318486
customersupport.uk@neb.com

For complex assemblies involving >10 fragments,
pre-cloned insert/modules levels can be decreased
from 75 to 50 ng each without significantly
decreasing the efficiencies of assembly.

10 Carefully design EVERY

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insert’s overhang

An assembly is only as good as its weakest
junction. Research at NEB has led to an increased
understanding of ligase fidelity, including thedevelopment of a comprehensive method forprofiling end-joining ligation fidelity in order topredict which overhangs will result in improved
accuracy. This ligase fidelity information can bepaired with the appropriate Type IIS restriction
enzyme chosen to direct your assembly to achieve
high efficiencies and accurate complex assemblies.
Please use the free NEBridge Golden Gate
Assembly Tool to design primers for your Golden
Gate Assembly reactions. Use NEBridge Ligase
Fidelity Tools to predict overhang fidelity or findoptimal Golden Gate junctions for long sequences.

f using pre-cloned proven
11 Iinsertsthat suddenly become

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problematic, check for a
possible mutational eventin your sequence
Be aware that occasionally a pre-cloned insert/module can become corrupted by an error during
propagation in E. coli, usually a frameshift due
to slippage in a run of a single base (e.g., AAAA)
by the E. coli DNA Polymerase. This should
be suspected if previously functional assemblycomponents suddenly become nonfunctional.

Featured AppNEBAR

Download the NEB AR App for iPhone®, iPad®
or Android™. Scan the augmented reality butterfly
icon located on the corner of the page to find videos,
tutorials and immersive experiences.

be INSPIRED
drive DISCOVERY
stay GENUINEFEATURED PRODUCTS:

With constant advances in both the development of new enzymes, tools and researchon maximizing enzyme functionality (e.g., ligase fidelity), NEB is the industry leader
in pushing the limits of Golden Gate Assembly and related methods.

PRODUCTNEB #SEQUENCE

BbsI

R0539S/LGAAGAC(2/6)

Try NEBridge Ligase Master Mix
for Added Flexibility
®

PaqCI

Type IIS restriction enzymes recognize asymmetric DNA sequences and cleave outsideof their recognition sequence. Type IIS enzymes commonly used in Golden Gate
Assembly are listed below. NEB currently offers over 50 Type IIS restriction enzymes.

5´CACCTGCNNNNNNNN  3´3´GTGGACGNNNNNNNN  5´Please visit www.neb.com for comprehensive table.

Advantages:

GAAGAC(2/6)

300/1,500 units

BsaI-HFv2

R3733S/L

GGTCTC(1/5)

1,000/5,000 units

BsmBI-v2

R0739S/L

CGTCTC(1/5)

200/1,000 units

BspQI

R0712S/L

GCTCTTC(1/4)

500/2,500 units

BtgZI

R0703S/L

GCGATG(10/14)

100/500 units

R0734S/L

CGTCTC(1/5)

300/1,500 units

R0745S/L

CACCTGC(4/8)

200/1,000 units

R0569S/L

GCTCTTC(1/4)

250/1,250 units

PaqCISapI• Assemble multiple fragments (2–50+)
in order, in a single reaction• Experience high efficiency, evenwith regions of high GC content
and areas of repeats• Use with a broad range of fragment
sizes (<100 bp to >15 kb)• Simplify reaction setup with oursuite of primer design and ligase
fidelity tools

GOLDEN GATE ASSEMBLY WORKFLOW
FOR BOTH SIMPLE AND COMPLEX ASSEMBLIES

p01

p03

p05

p04

NEB has developed a reliable set of enzymes anddesign tools for Golden Gate Assembly that we use
regularly with success. We have found the Ligase
Fidelity Viewer particularly useful for screeningoverhang sets that are constrained by a pre-existing
protein/DNA sequence. The thorough experimental
basis of the tool and the availability of theunderlying data are added bonuses.

PRODUCT

NEB #SIZE

NEBridge® Ligase Master Mix

M1100S/L 50/250 rxns

pGGAselect

CamR
gene

Can be used in complex (50+)
fragment assemblies

Correct assembly

The absence of internal sites in a sequence determines the choice of which
Type IIS restriction enzyme to drive the assembly. For your convenience,
NEB now offers two kits for Golden Gate Assembly featuring BsaI-HFv2or BsmBI-v2. Both kits incorporate digestion followed by ligation withT4 DNA Ligase into a single reaction, and can be used to assemble 2-50+
fragments in a single step.

10080

2x1066040

1x106

20NEBridge Ligase
T4 DNA
Master Mix
Ligase Buffer

0The total transformants and percentage of correct assemblies (blue colonies)
were reported as the average result of three replicates with the standard
deviation from the mean. The reaction with NEBridge Ligase Master Mix
generated 2.2 ± 0.2 x 10 6 correctly assembled blue colonies per µg vector
DNA with 94.3 ± 1% fidelity, while the reaction with T4 DNA Ligase Buffer
generated 8.3 ± 2.1 x 10 5 correctly assembled blue colonies per µg vector
DNA with 69.8 ±10.7% fidelity.

Ligase Fidelity Tools• 5 min. at 37°C (for BsaI-HFv2)
or 42°C (for BsmBI-v2)• 5 min. at 16°C (x 30–60 cycles)• 5 min. at 60°C

Incomplete assembly

A.

B.

PRODUCT

NEB #NEBridge Golden Gate
Assembly Kit (BsmBI-v2)

E1602S/L 20/100 rxns

NEBridge Golden Gate
Assembly Kit (BsaI-HFv2)

SIZE

E1601S/L 20/100 rxnsResearch at NEB has led to an increased understanding of ligase fidelity, including
the development of a comprehensive method for profiling end-joining ligation fidelity
to predict which overhangs have improved fidelity. This research has enabled thedevelopment of tools that enable the design of highly complex fragment assemblies
with high efficiencies and >90% accuracy.
Try our suite of free online tools to design high fidelity Golden Gate
Assemblies under various experimental conditions:...the Golden Gate Assembly paper: it revolutionizedthe technique.

ONE-POT GOLDEN GATE ASSEMBLYOF 52 FRAGMENTS INTO A DESTINATION VECTOR

Incorrect assemblyMix, incubate
and transform

Workflow for NEBridge Ligase Master Mix

0– Dr. Glenna Foight,Senior Scientist, Lyell Immunopharma

NEBridge Golden Gate Assembly Kits (BsmBI-v2 or BsaI-HF®v2)

p06

Single-tube reaction• BsaI-HFv2 or BsmBI-v2• T4 DNA Ligase

NEBridge™ Ligase
Master Mix

FEATURED KITS:

p02+Inserts

3x106• Perform single insert cloning in just
5 minutes using our fast protocols• Generate libraries with highefficiencies

Destinationvector

Blue = correctly
assembled colonies

CFU per µg Vector DNA

R3539S/L®

• BbsI-HF• BsaI-HFv2• BsmBI-v2/Esp3I• BspQI/SapI• PaqCI®

Plasmids or PCR-amplified DNA fragments®

300/1,500 units

BbsI-HF

Esp3I• Clone seamlessly, with no scars
remaining after assembly

What users are saying:

SIZENEBridge Ligase Master Mix is a 3X master mix for Golden Gate Assembly.
Designed for use with NEB Type IIS restriction enzymes, this master mix contains
T4 DNA Ligase in an optimized reaction buffer with a proprietary ligationenhancer. Users need only choose their preferred NEB Type IIS restriction
enzyme and add DNA substrates to be assembled. Low complexity single
fragment insertions, as well as moderate complexity (3–6 fragment) and highcomplexity (7–25+ fragment) assemblies, are all supported with this optimized
reagent and accompanying protocols.

Your choice of Type IIS
restriction enzyme:

Correct Assembly (%)

Push the limits ofGolden Gate AssemblyType IIS Restriction Enzymes used inGolden Gate Assembly

52 FRAGMENT lac ASSEMBLY COLONY FORMING UNITS*– Dr. Edward Green,
Team Leader, Cancer Research Center (DKZ)

For help designing primers, try the NEBridge Golden Gate
Assembly Tool at GoldenGate.neb.com
Try our NEBridge Ligase Fidelity Tools for the design ofhigh-fidelity Golden Gate assemblies at ligasefidelity.neb.com

CORRECTINCORRECT% CORRECT

Replicate #1

520

580

47

Replicate #2

760

740

51

• NEBridge Ligase Fidelity Viewer® (v2) – Visualize overhang ligation preferences

Replicate #3

900

880

51

• NEBridge GetSet® – Predict high-fidelity junction sets

Average72773349•N EBridge SplitSet® – Split DNA sequence for scarless high-fidelity assembly

Visit www.neb.com/GoldenGate
to learn more and view related videos
Golden Gate Assembly
Workflow
Golden Gate Assembly
Domestication
Golden Gate Assembly
Tool Tutorial
Listen to DAD whenconstructing high-complexity
Golden Gate Assembly Targets*per 100 µl of outgrowth platedBlue colony

How does
Golden Gate
Assembly work?

White colony[No colony]

In its simplest form, Golden Gate Assembly requires a Type IIS recognition site, added to bothends of a dsDNA fragment. After digestion, these sites are left behind, with each fragment bearing
the designed 3- or 4-base overhangs that direct the assembly.

(A) Example outgrowth plate used for colorimetric scoring by reverse blue-white screening. Correctly assembled 52 insert
constructs form blue colonies upon cellular transformation and incorrectly assembled constructs produce white colonies.
(B) Results of the assembly reactions. This replicate experiment was carried out to quantify the number of colony-forming
units harboring correct and incorrect assembly products per 100 μl of E. coli outgrowth plated (0.2 μl of the assembly
reaction). On average, 49% of the observed transformants harbored correctly assembled constructs.
Pryor, J. M. et al. (2022) ACS Synth. Biol., 11, 6, 2036–2042.

More information can be found in NEB publication, Comprehensive Profiling of Four
Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly(3), Enabling one-pot Golden Gate Assemblies of unprecedanted complexity using
data-optimized assembly design (4) or in our webinar, Listen to DAD Informatics
tools and NEB enzymes to enable complex one-pot Golden Gate Assemblies.

References
1. E ngler, C., Kandzia, R., and Marillonnet, S. (2008) PLoS ONE 3,e3647.
2. E
 ngler, C., et al. (2009) PLoS ONE 4, e5553.
3. P otapov, V. et. al. (2018) ACS Synth. Biol. 7,1, 2665–2674.
4. P ryor, J.M. et. al. (2020) PLoS ONE. 15: e0238592.