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Potvrzuji
  1. Úvodní strana
  2. Reagencie
  3. Life Technologies / Fermentas
  4. Conventional restriction enzymes
  5. SfiI

SfiI

Kód produktu: ER1821 Kód výrobce: ER1821 Kód dodavatele: {2DA659E6-1066-48A5-9696-935D80099678} Výrobce: Life Technologies Czech Republic s.r.o.
SfiI
1 730,30 Kč
1 430,00 Kč bez DPH
do týdne
1000 units

5'...G G C C N N N N↓N G G C C...3'
3'...C C G G N↑N N N N C C G G...5'

Zobraz detailní popis

Detailní popis

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.

Highlights

  • 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

Applications

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

Note

Assayed using pUC19 DNA with insert containing two SfiI sites from Adenovirus-2 DNA. Incubation at 37°C results in 10% activity. SfiI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam-, dcm- strain,  such as GM2163.

For cleavage with SfiI, at least two copies of its recognition sequence are required. The two sites can be on either the same or different DNA molecules. (L. M. Wertzell et al., J. Mol. Biol., 248, 581-595, 1995.) Therefore, also an oligonucleotide harboring a SfiI recognition site can be supplemented.

For methylation sensitivity refer to product specifications.

Conditions for 100% Activity
  • 1X Buffer G: 10 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, 50 mM NaCl, and 0.1 mg/mL BSA
  • Incubate at 50°C
Digestion of Agarose-embedded DNA Minimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded Adenovirus-2 DNA in 16 hours.
Double Digestion Perform double digestion using DoubleDigest.
Hazardous No
Isoschizomers Search for commercial isoschizomers using REsearch.
Storage Buffer SfiI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 300 mM NaCl, 10 mM MgCl2, 1 mM DTT, 0.15% 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)
1X		 G (green)
1X		 O (orange)
1X		 R (red)
1X		 Tango
(yellow)
1X / 2X
G (Green) 50°C 50-100 100 20-50 0-20 100 0-20 1X

Methylation Effects

  • Dam: never overlaps - no effect.
  • Dcm: may overlap - cleavage impaired.
  • CpG: may overlap - cleavage impaired.
  • EcoKI: never overlaps - no effect.
  • EcoBI: never overlaps - no effect.
Methylation type Sequence Cleavage effect
Dcm (CCWGG) 5'...GGCm5CW GGNNGGCC...3'
3'...CCG GWm5CCNNCCGG...5'		 Impaired
Dcm (CCWGG) 5'...Cm5CW GGCC(N)5GGCm5CW GG...3'
3'...G GWm5CCGG(N)5CCG GWm5CC...5'		 No effect
CpG 5'...m5C GGCC(N)5GGCm5C G...3'
3'... Gm5CCGG(N)5CCG Gm5C...5'		 No effect
CpG 5'...GGCm5C G(N)4GGCC...3'
3'...CCG Gm5C(N)4CCGG...5'		 Impaired
CpG 5'...GGCm5C G(N)3m5C GGCC...3'
3'...CCG Gm5C(N)3 Gm5CCGG...5'		 Blocked

Cleavage efficiency close to the termini of PCR fragments

bp from the recognition site to fragment end
1 2 3 4 5
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 0 0 0 0

New sites generated by ligation

Newly generated recognition sites resulting from removal of 3'-overhang and self-ligation

Recognition Sequence Newly generated sequence after reaction Restriction enzymes that cleave the newly generated recognition sequence
GGCCNNNN^NGGCC GGCCNNGGCC
  • BseDI (BsaJI)/FastDigest BsaJI (BseDI)
  • [BsuRI (HaeIII)/FastDigest HaeIII (BsuRI)]
  • [CviJI]

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