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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For more information about commercial rights, please contact NEB's Global Business Development team at firstname.lastname@example.org.
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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.
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.
Isothermal amplification generates many copies of a target sequence in a short period of time, at a constant temperature. Learn more about isothermal amplification.
Optical mapping is a method that allows production of restriction maps of whole chromosomes or genomes. Learn more about optical mapping.