cloning owl
  • My NEB
  • Print
  • PDF
  • Cloning & Synthetic Biology

    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.

    History of Cloning

    Download image as a PDF

    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

    Download image as a PDF

    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.

    Cloning & Synthetic Biology includes these areas of focus:

    DNA Preparation
    PCR
    Restriction Enzyme Digestion
    Reverse Transcription (cDNA Synthesis)
    Nucleic Acid Purification
    DNA End Modification
    Dephosphorylation
    Blunting
    Phosphorylation (Kinase)
    A-tailing
    DNA Assembly and Cloning
    NEBuilder® HiFi DNA Assembly
    Gibson Assembly®
    BioBrick® Assembly
    Golden Gate Assembly
    DNA Ligation
    Non-Cloning Ligation
    Cloning Ligation
    USER™ Cloning
    Transformation
    Genome Editing
    DNA Isolation
    DNA Analysis
    Restriction Enzyme Digestion
    Colony PCR
    DNA Sequencing
    Site Directed Mutagenesis

    FAQs for Cloning & Synthetic Biology

    Protocols for Cloning & Synthetic Biology