New to Cloning?

There are several methods and techniques available for cloning. Traditionally, cloning has utilized restriction enzymes to excise the DNA of interest, and to linearize a plasmid vector while creating compatible ends. After purification of the insert and vector, both are joined with the activity of a DNA ligase, and the newly-created recombinant vector is used to transform an E. coli host for propagation. PCR has also been used to generate both the vector and insert, which can be joined using a variety of techniques, ranging from standard DNA ligation or enzymatic joining using a recombinase or topoisomerase, to homologous recombination. More recently, DNA assembly techniques have been developed that utilize a mixture of enzymes to assemble DNA fragments in a single tube.

Learn more about the cloning workflow

 

Whether you are new to cloning, or having difficulties with an existing experiment, NEB offers a wide selection products, tools and resources that can help you be more efficient and successful with your experiments. To get started, choose the step in the cloning workflow below that you are interested in to find recommended products, videos, technical tips and more.

 

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DNA Assembly

Ordered assembly of multiple DNA fragments to create larger DNA structures has facilitated the field of synthetic biology. This application lends itself well to cloning of large or multiple fragments into a single vector. This technique is rapid and efficient, and can often be performed in a single tube.

 
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Nucleic acid purification

Purification of nucleic acids is an important part of the cloning workflow. Once plasmids containing a desired gene of interest are generated, they can be propagated using competent cells to increase the quantity of desired DNA. Plasmids then need to be recovered from their bacterial hosts using plasmid purification methods. It is important to separate plasmid DNA from the RNA and genomic DNA contained in host cells. Further, DNA must be concentrated and free from contaminating salts to achieve optimal enzyme activity. NEB’s Monarch® Nucleic Acid Purification kits enable quick and easy purification of high quality DNA for direct use in a variety of downstream applications.

 
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Restriction enzyme digestion

When performing traditional cloning, restriction enzyme (RE) sites that are unique to both the insert and vector should be chosen. Unidirectional cloning is achieved using two different restriction enzymes, each with unique recognition sites at the end of the insert. Depending on the RE chosen, ends can be blunt or sticky (cohesive). Restriction enzymes are generally used in traditional cloning, but are also needed for other DNA assembly approaches, such as NEBuilder® HiFi DNA Assembly, Gibson Assembly®, and Golden Gate Assembly. NEB offers the largest number of restriction enzyme specificities for your cloning experiments, and the largest number of specificities active in a single buffer, for simplified double digest reactions.

 
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PCR/amplification

Amplification can be performed to generate a blunt insert, or to have a 1-base overhang, depending on the polymerase used. Additionally, primers can be used to incorporate RE recognition sites. After amplification, the insert can be used directly or cloned into a holding vector, or RE digestion can be performed to generate cohesive ends. Amplification is often the first step for PCR cloning, seamless cloning, ligation independent cloning and recombinational cloning.

 
 

End modification

Phosphorylation: Vectors and inserts digested with restriction enzymes contain the necessary terminal modifications (5´ phosphate and 3´ hydroxyl) for ligation to occur. Typical amplification does not use phosphorylated primers, and therefore the 5´ ends will need to be treated with a kinase, such as T4 Polynucleotide Kinase (NEB #M0201).

Dephosphorylation: If a vector is linearized by a single restriction enzyme, or has been cut with two enzymes with compatible ends, a phosphatase is needed to remove the 5´ phosphate, reducing intramolecular ligation and therefore background during transformation. It is important to note that if a vector is dephosphorylated, the insert must contain a 5´ phosphate for ligation to occur.

Blunting/End Repair: Blunting is a process by which the single-stranded overhang created by an RE digest is either “filled in”, by adding nucleotides on the complementary strand using the overhang as a template for polymerization, or by “chewing back” the overhang, using an exonuclease. Vectors and inserts are often blunted to allow non-compatible ends to be joined.

A-tailing: Tailing is an enzymatic method for adding a non-templated nucleotide to the 3´ end of a blunt, dsDNA molecule. This is typically done to prepare a T-vector, or to A-tail a PCR product produced by a high fidelity polymerase for use in TA cloning. TA cloning is a rapid method of cloning PCR products that utilizes stabilization of the single-base extension produced by Taq DNA Polymerase by the complementary T of the T-vector prior to ligation and transformation. It is important to note that this method is non-directional and the insert can go into the vector in both orientations.

 
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DNA ligation

DNA ligation is commonly used in molecular cloning to join a DNA vector ("backbone") to a sequence of interest (“insert”). The ends of the DNA fragments can be blunt or cohesive, and at least one must contain a monophosphate group on its 5´ ends.

 
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Transformation

In cloning protocols, competent cells are used to introduce recombinant DNA into host bacteria. This transformation can be chemical, which utilizes divalent cations to increase cell membrane permeability, or electroporation can be employed, which utilizes electric current to create holes in the membrane. In either case, outgrowth of bacteria following transformation is required for propagation.

 
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DNA analysis

Agarose gel electrophoresis is the standard method used for separation, identification and purification of DNA fragments. DNA is visualized on a gel after soaking or pre-casting the gel with a visualization dye, such as ethidium bromide. DNA markers and ladders are composed of DNA fragments of known sizes and masses which are used as reference to determine the size and relative mass of the DNA of interest.

One or more of these products are covered by patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc. For more information, please email us at [email protected]. The use of these products may require you to obtain additional third party intellectual property rights for certain applications.

Feature Cloning Resources

ONLINE RESOURCES

NEBNext_portal_web_icon  Getting Started with Molecular Cloning
Explore simple tips to improve efficiency in your cloning experiment.
NEBNext_portal_web_icon  Troubleshooting Guide for Cloning
Find common problems and solutions associated with cloning experiments.
NEBNext_portal_web_icon  Traditional Cloning Quick Guide
Find quick tips for optimization of each step in the cloning workflow.

ONLINE TOOLS

tool_icon NEBcloner®
Find products, protocols and tips for each step of your traditional cloning experiment.
tool_icon NEBioCalculator®
For help with scientific calculations and conversions

BROCHURES & TECHNICAL GUIDES

NEBNext_portal_literature_icon Molecular Cloning Technical Guide
Get help with product selection, protocols, tips for optimization and trouble-shooting.
NEBNext_portal_literature_icon Reagents & Tools for Molecular Cloning Brochure
Learn about NEB's recommended products for cloning.