
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.
Choose Type:
- DNA Analysis
- Colony PCR
- DNA Sequencing
- Restriction Enzyme Digestion
- DNA Assembly and Cloning
- NEBuilder® HiFi DNA Assembly
- NEBridge® Golden Gate Assembly
- Gibson Assembly®
- BioBrick® Assembly
- DNA End Modification
- Dephosphorylation
- Phosphorylation (Kinase)
- A-tailing
- Blunting
- 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
- High-throughput cloning and automation solutions
- 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 DNA Assembly Electrocompetent Transformation Protocol
- NEBuilder® HiFi DNA Assembly Chemical Transformation Protocol (E2621, E5520, E2623)
- 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
- Transfection of EnGen® Spy Cas9 HF1 (NEB #M0667) into adherent cells using the Lipofectamine® RNAiMAX System
- 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
- Improved methods for site-directed mutagenesis using NEBuilder® HiFi DNA Assembly Master Mix
- Improved method for assembly of linear yeast expression cassettes using NEBuilder® HiFi DNA Assembly Master Mix
- Enhancing Transformation Efficiency
- Protocol for using Recombinant Cas9 Nuclease to Assess Locus Modification in Genome Editing Experiments
- Quick Ligation Kit
- Nanoliter Scale DNA Assembly Utilizing the NEBuilder HiFi Cloning Kit with the Labcyte Echo 525 Liquid Handler
- Fast & efficient isolation of phage genomic DNA using the Monarch HMW DNA Extraction Kits
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Restriction Endonucleases: Molecular Cloning and Beyond
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A Modern Day Gene Genie Sir Richard Roberts on Rebase
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How Gibson Assembly® is Changing Synthetic Biology
Understand how Gibson Assembly ® works and its impact in accelerating the progress of synthetic biology.
- Competent Cell Brochure
- DNA Assembly & Synthetic Biology
- Molecular Cloning Technical Guide
- NEBuilder HiFi DNA Assembly Bifold
- PCR Brochure
- NEBcloner®
- NEBioCalculator®
- PCR Selector
- Cleavage Of Supercoiled DNA
- Cloning Plasmids and DNAs
- Compatible Cohesive Ends and Generation of New Restriction Sites
- Competent Cell Selection Guide
- DNA Ligase Selection Chart
- DNA Markers & Ladders Selection Chart
- Dam-Dcm and CpG Methylation
- Frequencies of Restriction Sites
- 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
- 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
- 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
- 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), e00721-19. PubMedID: 31296691, DOI: 10.1128/MRA.00721-19
- 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
- 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
- Ke, Na; Berkmen, Mehmet; Ren, Guoping; (2017) A water-soluble DsbB variant that catalyzes disulfide-bond formation in vivo Nat Chem Biol; 13, 1022-1028. PubMedID: 28628094, DOI: 10.1038/nchembio.2409
- 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.
- 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, D298-D299. PubMedID: 25378308
- 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;
- 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), 11087-11092. PubMedID: 20129926
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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.
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