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Traditional Cloning
(Restriction Enzyme Cloning)
NEBuilder® HiFi
DNA Assembly
Golden Gate Assembly
Golden Gate Assembly is a single-tube reaction that uses Type IIS restriction enzymes and T4 DNA Lligase to assemble multiple inserts into a vector.
NEBuilder HiFi DNA Assembly enables fast and virtually error-free joining of DNA fragments with 15-30 bp overlaps, in a single-tube reaction.
Traditional Cloning usually refers to the use of restriction enzymes to generate DNA fragments with specific complementary ends that can be joined together with a DNA ligase, prior to transformation.
Advantages
disadvantages
Try NEBcloner® to receive product recommendations and protocols by answering some simple workflow questions
Learn more in our Overview of Traditional Cloning tutorial
Try NEBcloner
View Video Tutorial
Conceptually simple
Readily-available reagents in established labs
Scalable up to large amounts of DNA
Free online tools available
Time consuming
for many fragments
Requires unique RE sites in vector
and insert
Can leave
sequence scars behind
Advantages
Preferred option for new cloners
Single-tube reaction
Seamless
Fast
disadvantages
Requires
high-efficiency competent cells
Need to design primers & order separately
Not recommended for >11-fragment assemblies
Try NEBuilder Assembly Tool to design primers based upon fragment sequences and polymerase
Try NEBuilder Assembly Tool
View our application note on how NEBuilder is used to seamlessly assemble a vector expressing a fusion protein with multiple domains
View Application Note
Optimized mix available
Optimized for up
to 6 fragments
Can ultilize ssOligo assembly
Free online tool available
Possible
challenges with
sequence overlaps
Learn more in our Introduction to NEBuilder HiFi DNA Assembly tutorial
View Video Tutorial
Advantages
Single-tube reaction
Seamless
Fast
Use for complex assemblies – up to 50+ fragments
Tolerates areas
of repeats
Optimized mixes available
High efficiency
Free online tools available
disadvantages
Need to design primers & order separately
Type IIS enzyme site cannot be
present in vector*
Try NEBridge® Golden Gate Assembly Tool to design primers for your Golden Gate Assembly reactions
Try NEBridge Golden Gate Assembly Tool
View our publication on modular assembly for vector engineering
using Golden Gate Assembly
View Golden Gate Publication
Learn more in our introductory
Golden Gate Assembly Workflow tutorial
View Video Tutorial
Try NEBridge Ligase Fidelity Tools to design high-fidelity Golden Gate Assemblies under various conditions
Try NEBridge
Ligase Fidelity Tools
Find recommendations (i.e., insert:vector ratio and incubation time) for various scenarios using NEBuilder
View Usage Guidelines
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* Learn more about domestication in our tutorial
Type IIS enzyme site cannot be
present in vector*
Single-tube reaction
Seamless
Conceptually simple