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
AgeI §552
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
BamHI §555
BanI20205
BanII2055
EnzymeUnits for Complete Digestion
2 hours4 hours16 hours
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
EnzymeUnits for Complete Digestion
2 hours4 hours16 hours
BsmFI Δ2055
BsmI Δ**20
Bsp1286I552
BspEI2022
BspHI**(20)*(5)
BspMI**2
BsrBI**20
BsrDI Δ*202
BsrFI20202
BsrGI §222
BsrI Δ*52
BssHII Δ222
BstBI Δ522
BstEII  Δ §2055
BstNI Δ*52
BstUI Δ202020
BstXI552
BstYI Δ222
BstZ17I-HF®***(60)
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)
DraIII §2055
DrdI***
EnzymeBack to topUnits for Complete Digestion
2 hours4 hours16 hours
EaeI**20
EagI §552
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
EnzymeUnits for Complete Digestion
2 hours4 hours16 hours
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
MluI §522
MnlI2022
MscI*(20)*(5)*(5)
MseI2055
MslI**20
MspI***
MwoI Δ2022
EnzymeBack to topUnits for Complete Digestion
2 hours4 hours16 hours
NaeI2052
NarI222
EnzymeUnits for Complete Digestion
2 hours4 hours16 hours
NciI555
NcoI §552
NdeI**20
NgoMIV202020
NheI §20205
NheI-HF®20205
NlaIII**5
NlaIV2052
NotI §1022
NruI §222
NsiI §2055
EnzymeBack to topUnits for Complete Digestion
2 hours4 hours16 hours
PacI2052
PaeR7I522
PflMI**2
PleI2052
PmeI555
PmlI222
PpuMI555
PstI §552
PvuI §*2020
EnzymeUnits for Complete Digestion
2 hours4 hours16 hours
PvuII §202020
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
SacI §202020
SacII552
SalI §2052
SalI-HF®2052
SapI**20
Sau3AI2052
Sau96I20205
ScaI §***
ScaI-HF®***
ScrFI202020
SfaNI2552
SfcI**20
SfiI Δ2055
SmaI202020
SnaBI2022
SpeI §*(2)*(2)*(2)
EnzymeUnits for Complete Digestion
2 hours4 hours16 hours
SphI §***
SphI-HF®***
SspI §202020
StuI222
StyI §2052
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
(1) A HF version of this enzyme is available.
(2) A RE-Mix version of this enzyme is available.
HF® is a registered trademark of New England Biolabs, Inc.