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5'...CT C G A G...3'
3'...G A G C TC...5'

Available as a FastDigest enzyme for rapid DNA digestion

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

The XhoI restriction enzyme recognizes C^TCGAG sites and cuts best at 37°C in R buffer (Isoschizomers: PaeR7I, Sfr274I, SlaI, StrI, TliI)
Lambda DNA digested with XhoI

Lambda DNA digested with XhoI, 0.7% agarose, 1 cleavage site

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


For methylation sensitivity refer to product specifications.

Compatible EndsEco88I, PspXI, SmoI, SalI, SgrDI
Conditions for 100% Activity
  • 1X Buffer R: 10 mM Tris-HCl (pH 8.5 at 37°C), 10 mM MgCl2, 100 mM KCl, 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 lambda DNA in 16 hours.
Double DigestionPerform double digestion using DoubleDigest.
IsoschizomersSearch for commercial isoschizomers using REsearch.
NoteSupercoiled plasmids may require up to 5-fold more XhoI for complete digestion than linear DNA.
Storage BufferXhoI is supplied in 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 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
Red 37°C 0-20 50-100 50-100 100 20-50 100 1X or 2X

Methylation Effects

  • Dam: never overlaps - no effect.
  • Dcm: never overlaps - no effect.
  • CpG: completely overlaps - cleavage impaired.
  • EcoKI: never overlaps - no effect.
  • EcoBI: never overlaps - no effect.
Methylation type Sequence Cleavage effect
CpGCTCGAG Impaired

Cleavage efficiency close to the termini of PCR fragments

bp from the recognition site to fragment end
1 2 3 4 5
0-20 50-100

Number of recognition sites in DNA molecules

Lambda ΦX174 M13mp18/19 pBR322 puc18/19 pUC57
1 1 0 0 0 0
pTZ19R/U pTZ57R pBluescriptIIKS(-/+) pBluescriptIISK(-/+) pACYC177 pACYC184
0 0 1 1 1 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
  • AbsI (CC^TCGAGG)
  • Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (C^TCGAG)
  • PspXI* (VC^TCGAGC)
  • PspXI* (VC^TCGAGG)
  • PspXI* (VC^TCGAGT)
  • SmoI (SmlI)* (C^TCGAG)
  • BmeT110I
  • Eco88I (AvaI)/FastDigest AvaI (Eco88I)
  • SmoI (SmlI)
  • TaqI/FastDigest TaqI
  • XhoI/FastDigest XhoI
  • SalI/FastDigest SalI (G^TCGAC)
  • TaqI/FastDigest TaqI
  • Hpy99I
  • TaqI/FastDigest TaqI

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)
  • Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I)
  • DpnI/FastDigest DpnI
  • MboI/FastDigest MboI
  • PvuI/FastDigest PvuI
  • [TaqI/FastDigest TaqI]