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

Available as a FastDigest enzyme for rapid DNA digestion

FastDigest enzyme available Unique buffer for 100% activity Optimal incubation at 37°C Thermal inactivation at 65°C in 20 min Genome qualified LO certified

The SacI restriction enzyme recognizes GAGCT^C sites and cuts best at 37°C in its own unique buffer (Isoschizomers: Eco53kI, EcoICRI, Psp124BI, SstI)
Lambda DNA digested with SacI

Lambda DNA digested with SacI, 0.7% agarose, 2 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


SacI is sensitive to cytosine methylation at GAGmCTC, but not GAGCTmC and insensitive to adenine methylation at GmAGCTC. AluI methyltransferase (AGmCT) can be used to block SacI. Supercoiled plasmids may require up to 5-fold more SacI for complete digestion than linear DNA.

SacI is inhibited by common clinical anticoagulants found in some preparation of anticoagulated peripheral blood and bone marrow. Levels of EDTA and ACD (citric acid-sodium citrate-dextrose) in standard sample preparation have been shown to inhibit SacI. Three times the normal concentration for heparin is required to inhibit SacI. (J. E. Coad et al., Inhibition of restriction endonucleases by common clinical anticoagulants. Anal. Biochem. 205, 368-369, 1992).

For methylation sensitivity refer to product specifications.

Compatible EndsAlw21I, Eco24I, SduI.
Conditions for 100% Activity
  • 1X Buffer SacI: 10 mM Bis-Tris Propane-HCl (pH 6.5 at 37°C), 10 mM MgCl2, 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.
Storage BufferSacI 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
SacI Buffer (Unique) 37°C 50-100 20-50 0-20 0-20 50-100 20-50 1X or 2X

Methylation Effects

Methylation type Sequence Cleavage effect
CpG 5'...m5C GAGCTm5C G...3'
3'... Gm5CTCGA Gm5C...5'
No effect
EcoBI (TGA(N)8TGCT) 5'...TGm6AGCTC(N)4 TGCT...3'
3'...AC TCGAG(N)4m6ACGA...5'
No effect

Cleavage efficiency close to the termini of PCR fragments

bp from the recognition site to fragment end
1 2 3 4 5

Number of recognition sites in DNA molecules

Lambda ΦX174 M13mp18/19 pBR322 puc18/19 pUC57
2 0 1 0 1 1
pTZ19R/U pTZ57R pBluescriptIIKS(±) pBluescriptIISK(±) pACYC177 pACYC184
1 1 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
  • Alw21I (BsiHKAI)/FastDigest Alw21I* (GAGCT^C)
  • Eco24I (BanII)* (GAGCT^C)
  • SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GAGCT^C)
  • AluI/FastDigest AluI
  • Alw21I (BsiHKAI)/FastDigest Alw21I
  • CviJI
  • Ecl136II (EcoICRI)/FastDigest Ecl136II
  • Eco24I (BanII)
  • SacI/FastDigest SacI
  • SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)
  • SetI
  • SetI* (AGCT)
  • AluI/FastDigest AluI
  • CviJI
  • SetI