Traditional cloning by restriction endonuclease digestion can use any of a number of different source DNA types. Genomic DNA can be digested with a restriction enzyme and cloned into a compatible vector site to produce a library of different inserts, all from the same source DNA. DNA already cloned into one vector can be transferred (subcloned) to a new recipient vector by cutting out the DNA with restriction enzymes and cloning into the corresponding sites of the second vector. This is frequently undertaken to facilitate protein expression or transcription of RNA, for example, which might not be possible from the original vector.
PCR is often used for generating DNA for cloning and frequently restriction sites are incorporated into the primer sites so that the amplified DNA can be digested and cloned into compatible restriction sites of the cloning vector. Any type of DNA containing the desired sequence can serve as the template for PCR. Cloning with two distinct restriction enzymes ensures that non-compatible ends are generated on each molecule, thereby preventing simple vector recircularization and forcing inserts to be cloned directionally. This can be important for ensuring a translational open reading frame for protein expression. Modification of DNA ends following restriction digestion can be helpful in certain situations. For example, in non-directional cloning, where a single restriction enzyme is used, dephosphorylation of the digested vector DNA will prevent recircularization of the vector, thereby increasing the proportion of the desired recombinant DNA molecules.
PCR is increasingly used for preparing DNA for cloning applications. Amplified DNA can either be cloned directly, or following restriction digestion with restriction sites engineered into the primers used for PCR. Alternatively, amplified DNA can be used in Seamless Cloning strategies such as NEBuilder HiFi DNA Assembly or in Ligation Independent Cloning. The vector molecules for these cloning methods may also be produced by PCR. DNA amplified with Taq polymerase has a template-independent single adenosine (A) added at the 3’ ends which allows cloning into complementary T-tailed vectors. High-fidelity proofreading polymerases do not add additional bases allowing cloning of the amplified DNA into blunt-ended restriction sites. Depending on the chosen cloning strategy, the ends of amplified DNA can be modified by A-tailing, blunting, or addition or removal of 5’-phosphate groups. Complementary DNA (cDNA) generated by reverse transcription of RNA can also be amplified by PCR. This ability to synthesize DNA from RNA templates enables cloning of sequences corresponding to gene transcripts.
DNA Preparation includes these areas of focus:
Protocols for DNA Preparation
- Affinity Purification and On-column Cleavage (E6901)
- Cloning with USER Enzyme
- Comet Assay - Modified for Detection of Oxidized Bases Using the Repair Endonucleases Fpg, hOGG1 and Endonuclease III (Nth)
- Construction of the Fusion Plasmid (E6901)
- Double Digest Protocol with Standard Restriction Enzymes
- Fusion Constructs (E6901)
- Fusion Protein Expression (E6901)
- Optimizing Restriction Endonuclease Reactions
- Preparation of Media and Solutions (E6901)
- Primer Design for Restriction Enzyme Cloning (E6901)
- Protocol for Cre Recombinase (M0298)
- Protocol for Digestion Prior to droplet digital PCR (ddPCR)
- Protocol for Direct Digestion of gDNA during droplet digital PCR (ddPCR)
- Protocol for generating 32 bp fragments from modified CpG sites in genomic DNA using FspEI
- Protocol for generating 32 bp fragments from modified CpG sites in genomic DNA using MspJI
- Simplified Expression and Purification Protocol (E6901)
Molecular Cloning Technical Guide
The Molecular Cloning Technical Guide helps with product selection, protocols, tips for optimization and trouble-shooting.
Reagents and Tools for Molecular Cloning
Learn about recommended products for cloning in our Reagents and Tools for Molecular Cloning Brochure.
- A Modern Day Gene Genie Sir Richard Roberts on Rebase
- Why Choose the K. lactis Protein Expression Kit?
- Average Fragment Size Generated By Endonuclease Cleavage
- Compatible Cohesive Ends and Generation of New Restriction Sites
- Dam-Dcm and CpG Methylation
- Frequencies of Restriction Sites
- Recleavable Blunt Ends
- Recleavable Filled-in 5' Overhangs
- Why Choose Recombinant Enzymes?
- Troubleshooting Guide for Cloning
- Activity at 37°C for Restriction Enzymes with Alternate Incubation Temperatures
- Alteration of Apparent Recognition Specificities Using Methylases
- cDNA/Reverse Transcriptase Tips
- Cleavage Close to the End of DNA Fragments
- Cleavage of Supercoiled DNA
- Digestion of Agarose-Embedded DNA: Info for Specific Enzymes
- Double Digests
- Heat Inactivation
- Megabase Mapping
- NEBuffer Activity/Performance Chart with Restriction Enzymes
- Optimizing Restriction Endonuclease Reactions
- Reduced Star Activities of HF® Enzymes
- Restriction Endonucleases - Survival in a Reaction
- Restriction Enzyme Diluent Buffer Compatibility
- 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 DNA Preparation
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This overview will walk you through how the Polymerase Chain Reaction (PCR) works.
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.