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  • Restriction Enzyme Digestion

    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. 

    • Cloning With Restriction 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.

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    • Standard Protocol for Restriction Enzyme Digests

      Let one of NEB's restriction enzyme experts help you improve your technique and avoid common mistakes in digest setup.

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    • Why is My Restriction Enzyme Not Cutting DNA?

      Not getting the cleavage you expected? Let an NEB scientist help you troubleshoot your reaction.

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    • Restriction Enzyme Digest Problem: Too Many DNA Bands

      Are you finding unexpected bands in your digestion reaction? Here are some tips to help you determine the cause.

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    • What is Restriction Enzyme Star Activity?

      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.

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    • Setting-up Restriction Enzyme Digests with RE-Mix® Master Mixes

      RE-Mix® Restriction Enzyme Master Mixes offer simplified reaction setup. Learn more about digesting DNA with RE-Mix.

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    • RE-Mix® Double Digest Protocol

      Double digests are now easier than ever! Learn how to set up your next double digest with RE-Mix®.

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    • Reduce Star Activity with High-Fidelity Restriction Enzymes

      NEB has engineered HF™ enzymes to eliminate star activity. Learn how, and what this means for your digests.

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    • NEB Restriction Enzyme Double Digest Protocol

      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.

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    • Restriction Enzyme Digest Protocol: Cutting Close to DNA End

      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.

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    • Restriction Enzyme Digestion Problem: DNA Smear on Agarose Gel

      Learn more about what causes this common problem, and how NEB's enzymes are QC'd to avoid DNA smearing.

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    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.

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