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.
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.
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.
Cloning & Synthetic Biology includes these areas of focus:
- Fast Cloning: Accelerate your cloning workflows with reagents from NEB
- DNA Preparation
- Reverse Transcription (cDNA Synthesis)
- Restriction Enzyme Digestion
- Nucleic Acid Purification
- DNA End Modification
- Phosphorylation (Kinase)
- DNA Assembly and Cloning
- NEBuilder® HiFi DNA Assembly
- Gibson Assembly®
- BioBrick® Assembly
- Golden Gate Assembly
- DNA Ligation
- Non-Cloning Ligation
- Cloning Ligation
- USER® Cloning
- Applications of USER® and Thermolabile USER II Enzymes
- DNA Isolation
- DNA Analysis
- Restriction Enzyme Digestion
- Colony PCR
- DNA Sequencing
- Site Directed Mutagenesis
FAQs for Cloning & Synthetic Biology
Protocols for Cloning & Synthetic Biology
- Determining Genome Targeting Efficiency using T7 Endonuclease I
- Electroporation of Cas9 RNP (ribonucleoprotein) into adherent cells using the Neon® Electroporator
- In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)
- Insert Screening Protocols for NEB PCR Cloning Kit
- Ligation Protocol for NEB PCR Cloning Kit
- NEBuilder HiFi DNA Assembly Reaction Protocol
- NEBuilder® HiFi DNA Assembly Chemical Transformation Protocol
- NEBuilder® HiFi DNA Assembly Chemical Transformation Protocol (E2621)
- NEBuilder® HiFi DNA Assembly Electrocompetent Transformation Protocol
- NEBuilder® HiFi Electrocompetent Transformation Protocol (E2621)
- Plating Protocol for NEB PCR Cloning Kit
- RNA Synthesis of Cloned Insert Transcripts
- Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX
- Transformation Protocol for NEB PCR Cloning Kit
- Using recombinant Cas9 nuclease to assess locus modification in genome editing experiments (#M0386)
Application Notes Cloning & Synthetic Biology
- 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
A Modern Day Gene Genie Sir Richard Roberts on Rebase
Restriction Endonucleases: Molecular Cloning and Beyond
Competent Cells Brochure
The Competent Cells brochure provides information on the different competent cell strains for cloning and protein expression available from NEB.
The CutSmart™ Brochure provides information about the benefits of using CutSmart Buffer with NEB restriction enzymes.
DNA Ligase Brochure
The DNA Ligase brochure provides information about the extensive selection of DNA ligases and ligases master mixes available from NEB.
Molecular Cloning Technical Guide
Download the latest Molecular Cloning Technical Guide for help with product selection, protocols, tips for optimization and trouble-shooting.
NEBuilder HiFi DNA Assembly Bifold
Discover the next generation of gene assembly & cloning, and learn which advantages NEBuilder HiFi delivers over other gene assembly methods.
The PCR brochure provides product information on the wide range of DNA polymerases available from NEB, including tools for selection and troubleshooting tips.
Reagents & Tools for Molecular Cloning brochure
Learn about recommended products for cloning in our Reagents and Tools for Molecular Cloning Brochure.
Restriction Endonucleases Technical Guide
The Restriction Enzyme Technical Guide provides product information and technical reference charts on the wide range of restriction enzymes available from NEB.
Synthetic biology applies the framework of engineering and circuit theory to biological questions. Learn which products and protocols you'll need to get started.
- Prediction of Golden Gate Assembly GGA Using a Comprehensive Analysis of T4 DNA Ligase End-Joining Fidelity and Bias (2018)
- PCR Selector
- Average Fragment Size Generated By Endonuclease Cleavage
- 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
Other Tools & Resources
- 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.
Publications related to Cloning & Synthetic Biology
The first step in determining how your ends will be blunted is to determine if they are 5´ or 3´ overhangs. This tutorial will teach you how to identify what type of overhang you have, as well as which enzyme will blunt that end, and how.
Transformation is the process by which bacteria are made to take up exogenous DNA. The word is derived from Griffith's discovery of a "transforming principle". Learn more about transformation and how it is used in cloning workflows.
Phosphorylation is the process by which phosphate groups are added to a molecule by a kinase. The phosphorylation status of a fragment of DNA can influence its ability to proceed in reactions. Learn more about phosphorylation and kinases.
Dephosphorylation is the process by which phosphate groups are removed from a molecule by a phosphatase. Removal of phosphate groups from a DNA fragment can prevent ligation. Learn more about dephosphorylation and phosphatases.
What are toxic mini-genes, and how do they improve transformation efficiencies? Becky explains.
For the inside scoop on how NEB products come to be, learn the story behind the new NEB® PCR Cloning Kit.
PCR Cloning is an easy and reliable cloning method. The name is derived from the use of a DNA amplification step to generate the amplicon. Learn more about the benefits and disadvantages of PCR Cloning.
Restriction enzymes are an integral part of the cloning workflow, for generating compatible ends on fragments and vectors. This animation discusses three guidelines for determining which restriction enzymes to use in your cloning experiment.
Ligation, the process of joining DNA fragments with a DNA ligase, proceeds in three steps. Learn more about ligation with our quick animation.
Traditional Cloning refers to the generation of DNA fragments using restriction enzymes, and their subsequent assembly and transformation. The name is derived from the method’s history as the first widely-accepted cloning method. Learn more about the benefits and disadvantages of Traditional Cloning.