Preparation of DNA for traditional cloning methods is dependent upon restriction enzyme digestion to generate compatible ends capable of being ligated together. The DNA to be cloned can vary widely, from genomic DNA extracted from a pure bacterial culture or a mixed population, to a previously cloned gene that needs to be moved from one vector to another (subcloning). Restriction enzymes can also be used to generate compatible ends on PCR products. In all cases, one or more restriction enzymes are used to digest the DNA resulting in either non-directional or directional insertion into the compatible plasmid.
Genomic DNA, regardless of the source, is typically digested with restriction enzymes that recognize 6-8 consecutive bases, as these recognition sites occur less frequently in the genome than 4-base sites, and result in larger DNA fragments. The desired insert size for the clone library determines which enzymes are selected, as well as the digestion conditions. Most often, a serial dilution of the selected restriction enzyme(s) is used to digest the starting material and the desired insert size range is isolated by electrophoresis followed by gel extraction of the DNA. This method of preparation provides DNA fragments of the desired size with ends compatible to the selected vector DNA.
Subcloning requires the use of 1-2 restriction enzymes that cut immediately outside the insert fragment without cutting within the insert itself. Restriction enzymes that have a recognition site within the multiple cloning site (MCS) are commonly used since they do not cut elsewhere in the vector DNA and typically produce two easily resolved DNA fragments. The gene of interest is most commonly subcloned into an expression vector for improved protein expression and/or addition of a purification tag. In this case, it is essential that the gene be inserted in the correct orientation and in frame with the transcription promoter.
The Polymerase Chain Reaction (PCR) is commonly used to amplify a gene or DNA fragment of interest, from any source of DNA, to be cloned. In order to generate compatible ends, it is common to add restriction sites to the 5’ end of both PCR primers. When adding restriction sites to a PCR primer, it is recommended to include 6 bases between the recognition site and the 5’ end of the primer. These additional bases provide sufficient DNA for the restriction enzyme to bind the recognition site and cut efficiently. When selecting a restriction site(s) to add to the primers, it is important to determine which site(s) will be compatible with your selected vector, whether directional cloning is desired and, most importantly, confirm that the recognition site(s) does not occur within the gene or DNA fragment.
FAQs for Restriction Enzyme Digestion
Protocols for Restriction Enzyme Digestion
- Double Digest Protocol using One RE-Mix and One Standard Restriction Enzyme
- Double Digest Protocol using Two RE-Mix® Enzymes
- Double Digest Protocol with Standard Restriction Enzymes
- Optimizing Restriction Endonuclease Reactions
- 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 LpnPI (R0663)
- Protocol for generating 32 bp fragments from modified CpG sites in genomic DNA using MspJI
- Protocol for Glucosylation and digestion of Genomic DNA using AbaSI (#R0665)
- Removal of Single-Stranded Extension Protocol using Mung Bean Nuclease (M0250)
- Standard Digest Using RE-Mix®
The CutSmart™ Brochure provides information about the benefits of using CutSmart Buffer with NEB restriction enzymes.
Molecular Cloning Technical Guide
Download the latest Molecular Cloning Technical Guide for help with product selection, protocols, tips for optimization and trouble-shooting.
Reagents & Tools for Molecular Cloning brochure
Learn about recommended products for cloning in our Reagents and Tools for Molecular Cloning Brochure.
Restriction Endonucleases Technical Guide
The Restriction Enzyme Technical Guide provides product information and technical reference charts on the wide range of restriction enzymes available from NEB.
A Modern Day Gene Genie Sir Richard Roberts on Rebase
Everything You Ever Wanted to Know About Type II Restriction Enzymes
Restriction Endonucleases: Molecular Cloning and Beyond
Restriction Enzymes at NEB: Over 30 years of Innovation
Whole genome assembly from next generation sequencing data using restriction and nicking enzymes in optical mapping and proximity-based ligation strategies
High throughput sequencing methods have revolutionized genomic analysis by producing millions of sequence reads from an organism’s DNA at an ever decreasing cost.
- Alphabetized List of Recognition Specificities
- Average Fragment Size Generated By Endonuclease Cleavage
- Compatible Cohesive Ends and Generation of New Restriction Sites
- Cross Index of Recognition Sequences
- Dam-Dcm and CpG Methylation
- Enzymes with Multiple Recognition Sequences
- Enzymes with Nonpalindromic Sequences
- Frequencies of Restriction Sites
- Interrupted Palindromes
- Recleavable Blunt Ends
- Recleavable Filled-in 5' Overhangs
- Why Choose Recombinant Enzymes?
- Restriction Enzyme Troubleshooting Guide
- Troubleshooting Guide for Cloning
- Activity at 37°C for Restriction Enzymes with Alternate Incubation Temperatures
- Alteration of Apparent Recognition Specificities Using Methylases
- 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
- Restriction Enzyme Tips
- Restriction Enzymes for Droplet Digital PCR (ddPCR)
- Site Preferences
- Star Activity
- Traditional Cloning Quick Guide
Other Tools & Resources
- Fu YB, Peterson G. W., Dong Y 2016. Increasing Genome Sampling and Improving SNP Genotyping for Genotyping-by-Sequencing with New Combinations of Restriction Enzymes G3. 6:4, PubMedID: 26818077, DOI:
- 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.
- Loenen, W.A., Raleigh, E.A. 2014. The other face of restriction: modification-dependent enzymes. Nucleic Acids Res. 42, PubMedID: 23990325, DOI:
Publications related to Restriction Enzyme Digestion
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Type II restriction enzymes are most commonly used for molecular biology applications, as they recognize stereotypical sequences and produce a predictable cleavage pattern. Learn more about how Type II REs work.
Type I restriction enzymes are a group of endonucleases that recognize a bipartite sequence, but do not produce a predictable cleavage pattern. Learn more about how Type I REs work.
Type III restriction enzymes are a group of endonucleases that recognize a non-pallindromic sequence, comprising two inversely oriented sites. Learn more about these poorly understood enzymes.
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.
Let one of NEB's restriction enzyme experts help you improve your technique and avoid common mistakes in digest setup.
Not getting the cleavage you expected? Let an NEB scientist help you troubleshoot your reaction.
Are you finding unexpected bands in your digestion reaction? Here are some tips to help you determine the cause.
Learn what Star Activity is, why it is detrimental to accurate restriction enzyme digestion, and how NEB's HF enzymes are engineered to avoid it.
RE-Mix® Restriction Enzyme Master Mixes offer simplified reaction setup. Learn more about digesting DNA with RE-Mix.
Double digests are now easier than ever! Learn how to set up your next double digest with RE-Mix®.
NEB has engineered HF® enzymes to eliminate star activity. Learn how, and what this means for your digests.
Double digestions can save you time, and this video can offer tips for how to achieve the best results, no matter which of NEB's restriction enzymes you're using.
When cutting close to the end of a DNA molecule, make sure you know how many bases to add to the ends of your PCR primers.
Learn more about what causes this common problem, and how NEB's enzymes are QC'd to avoid DNA smearing.
Type IIS restriction enzymes have both recognition and binding sites, but cut downstream of the recognition site, creating 4-base overhangs ideal for reassembly. View a list of TypeIIS enzymes
Optical mapping is a method that allows production of restriction maps of whole chromosomes or genomes. Learn more about optical mapping.