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MspI (HpaII)

5'...CC G G...3'
3'...G G CC...5'

Available as a FastDigest enzyme for rapid DNA digestion

FastDigest enzyme available Tango buffer for 100% activity Optimal incubation at 37°C Thermal inactivation at 80°C in 20 min LO certified

The MspI (HpaII) restriction enzyme recognizes C^CGG sites and cuts best at 37°C in Tango buffer
Lambda DNA digested with MspI (HpaII)

Lambda DNA digested with MspI (HpaII), 1.4% agarose, 328 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


MspI is an isoschizomer of HpaII. When the external C in the sequence CCGG is methylated, MspI and HpaII cannot cleave. However, unlike HpaII, MspI can cleave the sequence when the internal C residue is methylated.

For methylation sensitivity refer to product specifications.

Compatible EndsBsp119I, Bsu15I, Hin1I, Hin6I, MaeII, NarI, Psp1406I, SsiI, TaqI, XmiI.
Conditions for 100% Activity
  • 1X Buffer Tango: 33 mM Tris-acetate (pH 7.9 at 37°C), 10 mM Mg-acetate, 66 mM K-acetate, and 0.1 mg/mL BSA
  • Incubate at 37°C
Double DigestionPerform double digestion using DoubleDigest.
IsoschizomersSearch for commercial isoschizomers using REsearch.
Storage BufferMspI is supplied in: 10 mM potassium phosphate (pH 7.5 at 25°C), 200 mM NaCl, 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
Tango 37°C 50-100 50-100 0-20 0-20 100 50-100 1X or 2X

Methylation Effects

Methylation type Sequence Cleavage effect
CpGCCGG No effect

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
328 5 18 26 13 13
pTZ19R/U pTZ57R pBluescriptIIKS(-/+) pBluescriptIISK(-/+) pACYC177 pACYC184
12 12 13 13 16 34

New sites generated by ligation

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

Recognition SequenceSecond EnzymeEnzymes recognizing newly generated recognition sequence
  • BmeT110I* (CY^CGGG)
  • BcnI (NciI)/FastDigest NciI (BcnI)
  • Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)
  • BssKI
  • HpaII/FastDigest HpaII
  • MspI (HpaII)/FastDigest MspI
  • Hin1I (BsaHI)/FastDigest BsaHI (Hin1I)* (GR^CGCC)
  • Hin6I (HinP1I)/FastDigest HinP1I (Hin6I) (G^CGC)
  • NarI (GG^CGCC)
  • SsiI (AciI)/FastDigest AciI (SsiI)* (C^CGC)
  • SsiI (AciI)/FastDigest AciI (SsiI)
  • HpaII/FastDigest HpaII (C^CGG)
  • SsiI (AciI)/FastDigest AciI (SsiI)* (G^CGG)
  • HpaII/FastDigest HpaII
  • MspI (HpaII)/FastDigest MspI

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

Recognition SequenceNewly generated sequence after reactionEnzymes that cleave the newly generated sequence
  • BseDI (BsaJI)/FastDigest BsaJI (BseDI)
  • Bsh1236I (BstUI)/FastDigest Bsh1236I
  • BtgI
  • Cfr42I (SacII)
  • MspA1I
  • SsiI (AciI)/FastDigest AciI (SsiI)