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:
  1. The DNA fragment of interest to be replicated
  2. 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. 

Plasmid vectors allow the DNA of interest to be copied in large amounts and, often, provide the necessary control elements to be used to direct transcription and translation of the cloned DNA. As such, they have become the workhorse for many molecular methods, such as protein expression, gene expression studies, and functional analysis of biomolecules.

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

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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

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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 Golden Gate 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.

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Cloning & Synthetic Biology includes these areas of focus:
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
Phosphorylation (Kinase)
DNA Ligation
Non-Cloning Ligation
Cloning Ligation
DNA Preparation
Reverse Transcription (cDNA Synthesis)
Restriction Enzyme Digestion
Fast Cloning: Accelerate your cloning workflows with reagents from NEB
Nucleic Acid Purification
Site Directed Mutagenesis
USER® Cloning
Applications of USER® and Thermolabile USER II Enzymes
FAQs for Cloning & Synthetic Biology
Protocols for Cloning & Synthetic Biology
Application Notes for Cloning & Synthetic Biology
    Publications related to Cloning & Synthetic Biology
  1. Gehring, A.M., Zatopek, K.M., Burkhart, B.W., Potapov, V., Santangelo, T.J., Gardner, A.F 2019. Biochemical reconstitution and genetic characterization of the major oxidative damage base excision DNA repair pathway in Thermococcus kodakarensis DNA Repair (Amst). , PubMedID: 31841800, DOI: 10.1016/j.dnarep.2019.102767
  2. Anton, B.P., Morgan, R.D., Ezraty, B., Manta, B., Barras, F., Berkmen, M. 2019. Complete genome sequence of Escherichia coli BE104, an MC4100 drivative lacking the methionine reductive pathway Microbiol Resour Announc. 8 (29), PubMedID: 31296691, DOI: 10.1128/MRA.00721-19
  3. Potapov, V., Ong, J.L., Kucera, R.B., Langhorst, B.W., Bilotti, K., Pryor, J.M., Cantor, E.J., Canton, B., Knight, T.F., Evans, T.C., Lohman, G.J.S. 2018. Comprehensive profiling of four base overhang ligation fidelity by T4 DNA ligase and application to DNA assembly ACS Synth Biol. 7 (11), PubMedID: 30335370, DOI: 10.1021/acssynbio.8b00333
  4. Ke, Na; Berkmen, Mehmet; Ren, Guoping; 2017. A water-soluble DsbB variant that catalyzes disulfide-bond formation in vivo Nat Chem Biol. 13, PubMedID: 28628094, DOI: 10.1038/nchembio.2409
  5. 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.
  6. Roberts, R.J., Vincze, T., Posfai, J., Macelis, D. 2015. REBASE - A database for DNA restriction and modification: enzymes, genes and genomes Nucleic Acids Res. 43, PubMedID: 25378308, DOI:
  7. Roberts, R.J., Vincze, T., Posfai, J., Macelis, D. 2014. REBASE - A database for DNA restriction and modification: enzymes, genes and genomes Nucleic Acids Res. , PubMedID: , DOI:
  8. 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:
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