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5'...G CG G C C G C...3'
3'...C G C C G GC G...5'

Available as a FastDigest enzyme for rapid DNA digestion

FastDigest enzyme available O buffer for 100% activity Optimal incubation at 37°C Cleavage blocked or impaired by CpG methylation Thermal inactivation at 80°C in 20 min Genome qualified High concentration available LO certified

The NotI restriction enzyme recognizes GC^GGCCGC sites and cuts best at 37°C in O buffer (Isoschizomers: CciNI)
Ad2 DNA digested with NotI

Ad2 DNA digested with NotI, 0.7% agarose, 7 cleavage sites

Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes, optimized to work in one of the buffers of the Five Buffer System. In addition, the universal Tango buffer is provided for convenience in double digestions. All of the enzymes exhibit 100% activity in the recommended buffer and reaction conditions. To ensure consistent performance, Thermo Scientific restriction enzyme reaction buffers contain premixed BSA, which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations.


  • Superior quality – stringent quality control and industry leading manufacturing process
  • Convenient color-coded Five Buffer System
  • Includes universal Tango buffer for double-digestions
  • BSA premixed in reaction buffers
  • Wide selection of restriction endonuclease specificities


  • Molecular cloning
  • Restriction site mapping
  • Genotyping
  • Southern Blot
  • Restriction fragment length polymorphism (RFLP)
  • SNP


Assayed using pTZ19RJL2 plasmid DNA digested with BseLI. Supercoiled plasmids may require up to 5-fold more NotI for complete digestion than linear DNAs.

For methylation sensitivity refer to product specifications.

Compatible EndsBsp120I, CfrI, Eco52I.
Conditions for 100% Activity
  • 1X Buffer O: 50 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, 100 mM NaCl, and 0.1 mg/mL BSA
  • Incubate at 37°C
Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded Adenovirus-2 DNA in 16 hours.
Double DigestionPerform double digestion using DoubleDigest.
IsoschizomersSearch for commercial isoschizomers using REsearch.
Storage BufferNotI is supplied in: 20 mM Tris-HCl (pH 7.8 at 25°C), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.02% Triton X-100, 0.2 mg/mL BSA, and 50% (v/v) glycerol.
Storage Condition-20 C

Reaction conditions

Recommended buffer for 100% activity Optimal temperature Enzyme activity in Thermo Scientific buffers, % Tango buffer for double digestion
B (blue)
G (green)
O (orange)
R (red)
1X / 2X
O (Orange) 37°C 0-20 20-50 100 20-50 0-20 20-50 2X

Methylation Effects

Methylation type Sequence Cleavage effect

Cleavage efficiency close to the termini of PCR fragments

bp from the recognition site to fragment end
1 2 3 4 5
20-50 50-100

Number of recognition sites in DNA molecules

Lambda ΦX174 M13mp18/19 pBR322 puc18/19 pUC57
0 0 0 0 0 0
pTZ19R/U pTZ57R pBluescriptIIKS(-/+) pBluescriptIISK(-/+) pACYC177 pACYC184
0 0 1 1 0 0

New sites generated by ligation

Newly generated recognition sites resulting from ligation of protruding compatible DNA ends

Recognition Sequence Second Enzyme Enzymes recognizing newly generated recognition sequence
  • Bsp120I (PspOMI)/FastDigest Bsp120I (G^GGCCC)
  • BmgT120I
  • BsuRI (HaeIII)/FastDigest HaeIII (BsuRI)
  • Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I)
  • CviJI
  • SatI (Fnu4HI)/FastDigest Fnu4HI (SatI)
  • TauI/FastDigest TauI
  • EaeI* (Y^GGCCA)
  • BsuRI (HaeIII)/FastDigest HaeIII (BsuRI)
  • EaeI
  • CviJI
  • SatI (Fnu4HI)/FastDigest Fnu4HI (SatI)
  • TauI/FastDigest TauI
  • EaeI* (Y^GGCCG)
  • Eco52I (EagI)/FastDigest EagI (Eco52I) (C^GGCCG)
  • Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I)
  • BsuRI (HaeIII)/FastDigest HaeIII (BsuRI)
  • EaeI
  • CviJI
  • Eco52I (EagI)/FastDigest EagI (Eco52I)
  • SatI (Fnu4HI)/FastDigest Fnu4HI (SatI)
  • TauI/FastDigest TauI

Newly generated recognition sites resulting from fill-in of 5'-overhang and self-ligation

Recognition Sequence Newly generated sequence after reaction Enzymes that cleave the newly generated sequence
  • [Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I)]
  • [BsuRI (HaeIII)/FastDigest HaeIII (BsuRI)]
  • Cac8I
  • Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)
  • [EaeI]
  • [CviJI]
  • [Eco52I (EagI)/FastDigest EagI (Eco52I)]
  • FseI
  • HpaII/FastDigest HpaII
  • MspI (HpaII)/FastDigest MspI
  • NgoMIV
  • PdiI (NaeI)/FastDigest NaeI (PdiI)
  • [SatI (Fnu4HI)/FastDigest Fnu4HI (SatI)]
  • [SsiI (AciI)/FastDigest AciI (SsiI)]
  • [TauI/FastDigest TauI]