Discover the efficiency and convenience of Thermo Scientific FastDigest restriction enzymes, designed to simplify your molecular biology workflows. FastDigest restriction enzymes help enable rapid DNA digestion in just 5–15 minutes, using a universal buffer system that helps ensure compatibility with all enzymes in the range. This helps streamline your processes, allowing for simultaneous digestion with multiple enzymes without the need for buffer changes. Explore our comprehensive selection of FastDigest restriction enzymes to help achieve accurate and reliable results in your cloning, sequencing, and other DNA manipulation experiments.

Watch the video for highlights of using FastDigest restriction enzymes over conventional restriction enzymes.

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     FastDigest restriction enzymesConventional restriction enzymes
    Buffer systemOne universal bufferUp to 20 buffers
    Double/multiple restriction digestionUnlimited—all enzymes are 100% active in one bufferLimited by buffer compatibility
    Reaction time5–15 minutes1 hour–overnight
    Direct loading on gelsYesNo
    Compatibility in downstream applications100%Partial
    Star activityNoYes
    Activity definition1 µL of FastDigest enzyme cleaves 1 µg of substrate DNA in 5 to 15 minutes in FastDigest buffer1 unit of enzyme hydrolyzes 1 µg of lambda DNA in 60 minutes in an optimal buffer for an enzyme


    FastDigest restriction enzyme buffers

    Reaction enzyme buffers help provide optimal conditions necessary for enzyme activity, including pH, ionic strength, and cofactors. They are crucial for precise DNA manipulation. A universal buffer for restriction enzymes is a significant advancement, simplifying experimental setups and reducing the need for multiple, specific buffers, thus helping save time and reducing errors.

    FastDigest reaction buffer is a specially formulated to support the activity of multiple different restriction enzymes, helping them to function efficiently in the same reaction mixture. The Thermo Scientific FastDigest system uses a universal buffer that is compatible with all FastDigest enzymes, which helps eliminate the need for buffer changes when using multiple enzymes, streamlining the digestion process and helping reduce the potential for experimental error.

    Universal restriction buffer: 100% activity in a single buffer

    The FastDigest Green Buffer and Thermo Scientific FastDigest Buffer are proprietary digestion buffers which help support 100% activity of all FastDigest restriction enzymes. This system allows for double and multiple digestions with any combination of enzymes (Figure 1). No sequential digestions or buffer changes are needed.

    DNA gel with clean, clear bands using FastDigest restriction enzymes in FastDigest buffer
    Figure 1. Five-minute plasmid DNA digestions with FastDigest restriction enzymes in FastDigest buffer. (Lane 1: Control, undigested plasmid DNA, lane 2: FastDigest EcoRI, lane 3: FastDigest EcoRI and FastDigest KpnI, lane 4: FastDigest EcoRI, FastDigest KpnI, and FastDigest SmaI, M: Thermo Scientific GeneRuler Express Ladder)

    FastDigest Buffer helps save time

    Because FastDigest buffers help support 100% activity of FastDigest restriction enzymes and do not require sequential digestions or buffer changes, protocol time is reduced. 

    Digestion times for reaction conditions are provided for all types of DNA templates (plasmid DNA, PCR product, genomic DNA) and no overnight digestions are required for any template. In addition, star activity is eliminated due to short reaction times. See an example of reaction times for a double digestion in Table 1.

    Table 1. Total reaction times for FastDigest restriction enzymes versus conventional restriction enzymes sequential digestion.

    Conventional restriction enzymes
    Sequential digestion    		FastDigest restriction enzymes
    One reaction mixture
    Reaction setup for Apal~2 minReaction set up with FastDigest ApaI and XhoI~2 min
    Incubation60 minIncubation5 min
    Reaction setup for Xhol~2 min——
    Incubation60 min——
    Total time>2 hoursTotal time 7 min

    FastDigest buffer compatibility with downstream applications

    DNA/RNA modifying enzymes, such as ligases, phosphatases, kinases, and mesophilic DNA polymerases have up to 100% activity in FastDigest Buffer and FastDigest Green Buffer (Table 2). Therefore, enzymes used in downstream applications can be directly added to the FastDigest reaction mix. No buffer changes or purification steps are needed.

    Table 2. Percent activity of DNA/RNA modifying enzymes in FastDigest buffers.

    DNA/RNA modifying enzymeActivity in FastDigest Green Buffer/FastDigest Buffer 
    DNA Polymerase I (10 U/μL) product photoDNA Polymerase I, E. coli100%Order now
    Klenow Fragment (10 U/μL) product photoKlenow Fragment100%Order now
    Klenow Fragment, exo– (5 U/μL) product photoKlenow Fragment, exo-100%Order now
    T4 DNA Polymerase (5 U/μL) product photoT4 DNA Polymerase100%Order now
    T7 DNA Polymerase (10 U/μL) product photoT7 DNA Polymerase  100%Order now
    T4 DNA Ligase (5 U/μL) product photoT4 DNA Ligase*75–100%Order now
    FastAP Thermosensitive Alkaline Phosphatase (1 U/μL) product photoFastAP Thermosensitive Alkaline Phosphatase100%Order now
    T4 Polynucleotide Kinase (10 U/μL) product photoT4 Polynucleotide Kinase100%Order now

    *0.5 mM ATP is required for T4 DNA Ligase activity

    FastDigest Green Buffer contains two tracking dyes

    FastDigest Green Buffer offers the same performance as the colorless FastDigest Buffer, but FastDigest Green Buffer is ready for direct loading of the reaction mixture into gels. The 10X FastDigest Green Buffer includes a density reagent and two tracking dyes for direct loading. In a 1% agarose gel, the blue dye migrates with 3–5 kb DNA fragments and has an excitation peak of 424 nm, while the yellow dye migrates faster than 10 bp DNA fragments and has an excitation peak of 615 nm (Figure 2).

    A schematic of FastDigest Green buffer: how it looks before and after electrophoresis, with the latter separating into blue and yellow bands at distinct bp lengths
    Figure 2. FastDigest Green buffer contains two tracking dyes: blue and yellow. Lane 1 is the reaction mixture in FastDigest Green Buffer loaded into a gel well before electrophoresis. Lane 2 is the reaction mixture in FastDigest Green Buffer separated after electrophoresis. The blue dye migrates around 3–5 kb and the yellow dye migrates around 10 bp.

    FastDigest enzymes with FastDigest Green Buffer designed to help eliminate star activity

    FastDigest restriction enzymes in combination with FastDigest Green Buffer are designed to eliminate star activity due to short incubation time and optimal enzyme and glycerol concentrations.

    Conventional restriction enzymes may display star or "relaxed" activity due to prolonged incubation times, high enzyme and/or glycerol concentration, high pH values, or low ionic strength. By addressing all these issues, FastDigest enzymes enable single, double and even triple digestion in five minutes without any signs of star activity.


    References using FastDigest enzymes and buffers

    ApplicationsPublications
    FastDigest EcoRI and BamHI restriction enzymes were used to prepare vector for Golden Gate cloning methodWang T, Wang D, Lyu Y, et al. (2018) Construction of a high-efficiency cloning system using the Golden Gate method and I-SceI endonuclease for targeted gene replacement in Bacillus anthracis. J Biotechnol 271:8–16. 
    FastDigest BpiI (BbsI) Type IIS restriction enzyme was used to construct gRNAKurata M, Wolf NK, Lahr WS, et al. (2018) Highly multiplexed genome engineering using CRISPR/Cas9 gRNA arrays. PLoS ONE 13(9):e0198714. 
    FastDigest TaaI restriction enzyme was used in RFLP analysis of PCR products
    		Fong WY, Ho CC, Poon WT. (2017) Comparison of Direct Sequencing, Real-Time PCR-High Resolution Melt (PCR-HRM) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis for Genotyping of Common Thiopurine Intolerant Variant Alleles NUDT15 c.415C>T and TPMT c.719A>G (TPMT*3C). Diagnostics (Basel) 7(2):27. 
    Fragmentation of genomic DNA with FastDigest HindIII restriction enzymes prior to ddPCRLi B, Kaushik S, Kalinowski P, et al. (2018) Droplet digital PCR shows the D-Loop to be an error prone locus for mitochondrial DNA copy number determination. Sci Rep 8(1):11392. 

    Resources for FastDigest restriction enzymes

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    For Research Use Only. Not for use in diagnostic procedures.

    Stylesheet for Classic Wide Template adjustments