
Cloning & Synthetic Biology
Clone with Confidence™
Molecular cloning refers to the process by which recombinant DNA molecules are produced and transformed into a host organism, where they are replicated. A molecular cloning reaction is typically comprised of the following two components:- The DNA fragment of interest to be replicated
- A vector/plasmid backbone that contains all of the components for replication in the host
DNA of interest, such as a gene, regulatory element(s), or operon, etc., is prepared for cloning by excising it out of the source DNA using restriction enzymes, copying it using the Polymerase Chain Reaction (PCR), or assembling it from individual oligonucleotides. At the same time, a plasmid vector is prepared in linear form using restriction enzymes or PCR. The plasmid is a small, circular piece of DNA that is replicated within the host, and exists separately from the host’s chromosomal or genomic DNA. By physically joining the DNA of interest to the plasmid vector through phosphodiester bonds, the DNA of interest becomes part of the new recombinant plasmid and is replicated by the host.
During the cloning process, the ends of the DNA of interest and the vector have to be modified to make them compatible for joining through the action of a DNA ligase, recombinase, or in vivo DNA repair mechanism. These steps typically utilize enzymes, such as nucleases, phosphatases, kinases and/or ligases. Many cloning methodologies and, more recently, kits have been developed to simplify and standardize these processes.
Use NEBcloner to find the right products and protocols for each cloning step.
History of Cloning

Learn more about the various types of molecular cloning found in the workflow below: Traditional Cloning, PCR Cloning, Seamless Cloning, Ligation Independent Cloning (LIC) and Recombinational Cloning.
Cloning Workflow

Synthetic Biology
Synthetic Biology is a more recent expansion of the biotechnology field, in which genes and proteins are viewed as parts or devices, with the goal of re-designing and/or assembling these parts in novel ways to create a new and useful functionality. Recent advances in biofuels generation, production of biochemicals, and understanding the minimal genome all benefit from synthetic biological approaches. Often these projects rely on the ordered assembly of multiple DNA sequences to create large, artificial DNA structures. To this end, methods have evolved to simplify this process. NEBuilder® HiFi DNA Assembly and Gibson Assembly® can be used to create many functional DNA structures, from a simple joining of two metabolic genes, all the way up to the creation of an artificial genome.
To help select the best DNA assembly method for your needs, please use our Synthetic Biology/DNA Assembly Selection Chart.
Learn more at NEBuilderHiFi.com.
Choose Type:
- DNA Analysis
- Colony PCR
- DNA Sequencing
- Restriction Enzyme Digestion
- DNA Assembly and Cloning
- NEBuilder® HiFi DNA Assembly
- Gibson Assembly®
- BioBrick® Assembly
- Golden Gate Assembly
- DNA End Modification
- Dephosphorylation
- Phosphorylation (Kinase)
- A-tailing
- Blunting
- DNA Isolation
- DNA Ligation
- Non-Cloning Ligation
- Cloning Ligation
- DNA Preparation
- Reverse Transcription (cDNA Synthesis)
- Restriction Enzyme Digestion
- PCR
- Fast Cloning: Accelerate your cloning workflows with reagents from NEB
- Nucleic Acid Purification
- Site Directed Mutagenesis
- Transformation
- USER® Cloning
- Applications of USER® and Thermolabile USER II Enzymes
- Insert Screening Protocols for NEB PCR Cloning Kit
- Ligation Protocol for NEB PCR Cloning Kit
- Plating Protocol for NEB PCR Cloning Kit
- Transformation Protocol for NEB PCR Cloning Kit
- In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)
- Determining Genome Targeting Efficiency using T7 Endonuclease I
- NEBuilder HiFi DNA Assembly Reaction Protocol
- NEBuilder® HiFi Electrocompetent Transformation Protocol (E2621)
- NEBuilder® HiFi DNA Assembly Electrocompetent Transformation Protocol
- NEBuilder® HiFi DNA Assembly Chemical Transformation Protocol (E2621)
- NEBuilder® HiFi DNA Assembly Chemical Transformation Protocol
- Using recombinant Cas9 nuclease to assess locus modification in genome editing experiments (#M0386)
- Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX
- RNA Synthesis of Cloned Insert Transcripts
- Genomic DNA Cleanup Protocol
- Improved method for assembly of linear yeast expression cassettes using NEBuilder® HiFi DNA Assembly Master Mix
- Improved methods for site directed mutagenesis using NEBuilder HiFi DNA Assembly Master Mix
- Improved methods for site-directed mutagenesis using Gibson Assembly Master Mix
- Robust Colony PCR from Multiple E. coli Strains using OneTaq® Quick-Load® Master Mixes
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A Modern Day Gene Genie Sir Richard Roberts on Rebase
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Restriction Endonucleases: Molecular Cloning and Beyond
- Competent Cells Brochure
- CutSmart™ Brochure
- DNA Ligase Brochure
- Molecular Cloning Technical Guide
- NEBuilder HiFi DNA Assembly Bifold
- PCR Brochure
- Reagents & Tools for Molecular Cloning brochure
- Restriction Endonucleases Technical Guide
- Synthetic Biology
- NEBcloner
- NEBioCalculator
- PCR Selector
- Cloning Plasmids and DNAs
- Compatible Cohesive Ends and Generation of New Restriction Sites
- Competent Cell Selection Guide
- Dam-Dcm and CpG Methylation
- DNA Ligase Selection Chart
- DNA Markers & Ladders Selection Chart
- Frequencies of Restriction Sites
- PCR Selection Tool
- Recleavable Blunt Ends
- Recleavable Filled-in 5' Overhangs
- Synthetic Biology/DNA Assembly Selection Chart
- Why Choose Recombinant Enzymes?
- PCR Troubleshooting Guide
- Troubleshooting Guide for Cloning
- Troubleshooting Tips for Ligation Reactions
- Activity at 37°C for Restriction Enzymes with Alternate Incubation Temperatures
- Chemical Transformation Tips
- Cleavage Close to the End of DNA Fragments
- Cleavage Of Supercoiled DNA
- Digestion of Agarose-Embedded DNA: Info for Specific Enzymes
- Double Digests
- Electroporation Tips
- Getting Started with Molecular Cloning: Simple Tips to Improve your Cloning Efficiency
- Optimizing Restriction Endonuclease Reactions
- Reduced Star Activities of HF® Enzymes
- Restriction Endonucleases - Survival in a Reaction
- Site Preferences
- Star Activity
- Traditional Cloning Quick Guide
- Prediction of Golden Gate Assembly GGA Using a Comprehensive Analysis of T4 DNA Ligase End-Joining Fidelity and Bias (2018)
Feature Articles
Brochures
Web Tools
Selection Tools
Troubleshooting Guides
Usage Guidelines
Posters
- Shah, S., Sanchez, J., Stewart, A., et al. 2015. Probing the Run-On Oligomer of Activated SgrAI Bound to DNA PLoS One. 10(4), PubMedID: 25880668, DOI: 10.1371/journal.pone.0124783.
- Mauris, J.and Evans, T.C., Jr. 2010. A human PMS2 homologue from Aquifex aeolicus stimulates an ATP-dependent DNA helicase. J Biol.Chem. 285(15), PubMedID: 20129926, DOI:
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].
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.