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AlfI

5'...↓10(N) G C A (N)6 T G C (N)12↓...3'
3'...↑12(N) C G T (N)6 A C G (N)10↑...5'

Genome qualified Optimal incubation at 37°C LO certified R buffer for 100% activity Requires SAM for stated activity Thermal inactivation at 65°C in 20 min

The AlfI restriction enzyme recognizes ^(10/12)GCA(N)6TGC(12/10)^ sites and cuts best at 37°C in R (+SAM) buffer
  
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Lambda DNA digested with AlfI

Lambda DNA digested with AlfI, 1.0% agarose, 22 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.

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

For methylation sensitivity refer to product specifications..

  
Storage Condition-20 C
HazardousNo
Conditions for 100% Activity
  • [1X Buffer R] + SAM: [10 mM Tris-HCl (pH 8.5 at 37°C), 10 mM MgCl2, 100 mM KCl and 0.1 mg/mL BSA] + 0.01 mM S-adenosylmethionine.
  • 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.
NoteComplete cleavage of some substrates with AlfI is difficult to achieve. AlfI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.
Storage BufferAlfI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/mL BSA and 50% (v/v) glycerol.

Reaction conditions

Recommended buffer for 100% activity Optimal tempThermo Scientificure 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
R (red) +SAM 37°C 0-20 (+SAM) 0-20 (+SAM) 0-20 (+SAM) 100 (+SAM) 0-20 (+SAM) 20-50 (+SAM) 2X (+SAM)

Methylation Effects

  • Dam: never overlaps - no effect.
  • Dcm: never overlaps - no effect.
  • CpG: may overlap - no effect.
  • EcoKI: never overlaps - no effect.
  • EcoBI: never overlaps - no effect.
Methylation type Sequence Cleavage effect
CpG5'...GCA(N)6TGm5C G...3'
3'...CGT(N)6AC Gm5C...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
0

Number of recognition sites in DNA molecules

Lambda ΦX174 M13mp18/19 pBR322 puc18/19 pUC57
22 0 1 3 0 1
pTZ19R/U pTZ57R pBluescriptIIKS(±) pBluescriptIISK(±) pACYC177 pACYC184
0 1 0 0 1 3