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Home | Molecular Biology | Restriction & Modifying Enzymes | Restriction Enzymes | Conventional

Alw44I

5'...G↓T G C A C...3'
3'...C A C G T↑G...5'
Available as a FastDigest enzyme for rapid DNA digestion

FastDigest enzyme available Tango buffer for 100% activity Optimal incubation at 37°C Cleavage blocked or impaired by overlapping CpG methylation Thermal inactivation at 65°C in 20 min Genome qualified LO certified

The Alw44I (ApaLI) restriction enzyme recognizes G^TGCAC sites and cuts best at 37°C in Tango buffer (Isoschizomers: ApaLI, VneI)
  
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Lambda DNA digested with Alw44I

Lambda DNA digested with Alw44I, 0.7% agarose, 4 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
Compatible EndsBfmI
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
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 BufferAlw44I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 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 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
Tango 37°C 50-100 100 0-20 50-100 100 50-100 1X or 2X

Methylation Effects

Methylation type Sequence Cleavage effect
CpG 5'...GTGCAm5C G...3'
3'...CACGT Gm5C...5'		 Blocked
EcoKI (AAC(N)6GTGC) 5'...Am6AC(N)6G TGCAC...3'
3'...T TG(N)6Cm6ACGTG...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 20-50 50-100

Number of recognition sites in DNA molecules

Lambda ΦX174 M13mp18/19 pBR322 puc18/19 pUC57
4 1 1 3 3 3
pTZ19R/U pTZ57R pBluescriptIIKS(±) pBluescriptIISK(±) pACYC177 pACYC184
2 2 2 2 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
G^TGCAC
  • BfmI (SfcI)/FastDigest SfcI (BfmI)* (C^TGCAG)
  • BsgI
  • HpyCH4V

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
G^TGCACGTGCATGCAC
  • Cac8I
  • CviAII
  • FaiI
  • FatI
  • Hin1II (NlaIII)/FastDigest NlaIII (Hin1II)
  • [HpyCH4V]
  • PaeI (SphI)/FastDigest SphI (PaeI)
  • XceI (NspI)/FastDigest NspI (XceI)