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  • Digestion of Agarose-Embedded DNA: Info for Specific Enzymes

    Restriction endonuclease digestion of agarose-embedded DNA has proven to be a valuable tool in the physical mapping of chromosomes. To compare restriction endonuclease performance under controlled conditions, purified substrate DNAs (lambda, adenovirus-2) were embedded in 1% ImBed® low-melting-point agarose and subsequently digested. The results are presented below.

    Substrate DNAs were embedded in 1% ImBed agarose (1 µg DNA/30 µl). The GelSyringe™ agarose- dispensing system was used to produce 30 µl agarose plugs. The agarose plugs were equilibrated in the recommended 1X NEBuffer by two 15 minute washes (100 µl each) and then incubated in 100 µl of fresh 1X NEBuffer plus 2, 5, or 20 units of enzyme for 2, 4 or 16 hours (total reaction volume of 130 µl). Most enzyme reactions were incubated at 37°C; thermophilic enzymes (D) were incubated at 50°C. After digestion, cold TE [10 mM Tris-HCl, 0.5 mM EDTA] was used to stop the reactions. The resulting digests were resolved on 1% agarose gels and compared to a standard digest (1 µg, 50 µl liquid reaction volume, 1 hour).

    The values reported are the minimum units required for complete digestion. An asterisk (*) indicates that digestion was incomplete at that time point with the maximum number of units used in these tests (20 units). In some cases, the experiment was repeated after a 16 hour preincubation (at 4°C) to help diffuse the enzyme into the plug. Values in parentheses indicate the minimum units required for complete digestion after preincubation. It should also be noted that the actual number of units required for complete digestion will vary depending on the DNA substrate and its level of purity.

    Limited enzyme diffusion into the agarose matrix may account for diminished enzyme performance on agarose-embedded DNA. To reduce this effect, plugs should be made with low-melting-point agarose at concentrations of 0.5-1.0%. A 16 hour preincubation of the reaction (at 4°C) is useful in cases where enzyme stability is compromised by incubation at 37°C for extended periods of time. Gentle mixing of the reaction has also been reported to aid in the diffusion of enzyme into the plug.

    EnzymeUnits for Complete Digestion
    2 hours4 hours16 hours
    AatII202020
    AccI522
    AciI**(20)*(20)
    AflII222
    AflIII**2
    AgeI552
    AluI*2020
    AlwI**2
    AlwNI*2020
    ApaI*(20)*(20)*(20)
    ApaLI222
    ApoI Δ*2020
    AscI*202
    AseI*52
    AvaI**(20)20
    AvaII222
    AvrII*22
    EnzymeBack to topUnits for Complete Digestion
    2 hours4 hours16 hours
    BamHI555
    BanI20205
    BanII2055
    BbsI222
    BbvI**5
    BcgI*55
    BclI Δ***
    BfaI202020
    BglI**5
    BglII**2
    BpmI**20
    BsaAI**20
    BsaBI Δ**20
    BsaHI2052
    BsaI***
    BsaJI Δ*205
    BsaWI Δ*202
    BsgI2055
    BsiEI Δ522
    BsiHKAI Δ222
    BsiWI Δ20202
    BslI Δ*55
    BsmAI Δ**2
    BsmFI Δ2055
    BsmI Δ**20
    Bsp1286I552
    BspEI2022
    BspHI**(20)*(5)
    BspMI**2
    BsrBI**20
    BsrDI Δ*202
    BsrFI20202
    BsrGI222
    BsrI Δ*52
    BssHII Δ222
    BstBI Δ522
    BstEII  Δ2055
    BstNI Δ*52
    BstUI Δ202020
    BstXI552
    BstYI Δ222
    Bsu36I*52
    EnzymeBack to topUnits for Complete Digestion
    2 hours4 hours16 hours
    ClaI2055
    EnzymeBack to topUnits for Complete Digestion
    2 hours4 hours16 hours
    DdeI2052
    DpnI*(20)*(5)*(5)
    DpnII222
    DraI*(20)*(20)*(20)
    DraIII2055
    DrdI***
    EnzymeBack to topUnits for Complete Digestion
    2 hours4 hours16 hours
    EaeI**20
    EagI552
    EarI20205
    EcoNI202020
    EcoO109I***
    EcoRI**20
    EcoRV*202
    EnzymeBack to topUnits for Complete Digestion
    2 hours4 hours16 hours
    FokI2022
    FspI20102
    EnzymeBack to topUnits for Complete Digestion
    2 hours4 hours16 hours
    HaeII20202
    HaeIII202020
    HgaI*2020
    HhaI20202
    HincII**2
    HindIII*2020
    HinfI*22
    HinP1I2052
    HpaI*2020
    HpaII552
    HphI2052
    EnzymeBack to topUnits for Complete Digestion
    2 hours4 hours16 hours
    KasI***
    KpnI*2020
    EnzymeBack to topUnits for Complete Digestion
    2 hours4 hours16 hours
    MboI222
    MboII222
    MluI522
    MnlI2022
    MscI*(20)*(5)*(5)
    MseI2055
    MslI**20
    MspI***
    MwoI Δ2022
    EnzymeBack to topUnits for Complete Digestion
    2 hours4 hours16 hours
    NaeI2052
    NarI222
    NciI555
    NcoI552
    NdeI**20
    NgoMIV202020
    NheI20205
    NheI-HF®20205
    NlaIII**5
    NlaIV2052
    NotI1022
    NruI222
    NsiI2055
    EnzymeBack to topUnits for Complete Digestion
    2 hours4 hours16 hours
    PacI2052
    PaeR7I522
    PflMI**2
    PleI2052
    PmeI555
    PmlI222
    PpuMI555
    PstI552
    PvuI*2020
    PvuII202020
    PvuII-HF®202020
    EnzymeBack to topUnits for Complete Digestion
    2 hours4 hours16 hours
    RsaI202020
    RsrII1052
    EnzymeBack to topUnits for Complete Digestion
    2 hours4 hours16 hours
    SacI202020
    SacII552
    SalI2052
    SalI-HF®2052
    SapI**20
    Sau3AI2052
    Sau96I20205
    ScaI***
    ScaI-HF®***
    ScrFI202020
    SfaNI2552
    SfcI**20
    SfiI Δ2055
    SmaI202020
    SnaBI2022
    SpeI*(2)*(2)*(2)
    SphI-HF®***
    SphI***
    SspI202020
    StuI222
    StyI2052
    EnzymeBack to topUnits for Complete Digestion
    2 hours4 hours16 hours
    TaqαI Δ***
    TfiI Δ*202
    Tsp509I Δ**20
    Tth111I Δ**5
    EnzymeBack to topUnits for Complete Digestion
    2 hours4 hours16 hours
    XbaI*52
    XcmI**20
    XhoI202020
    XmaI*52
    XmnI**5