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Acc65I (Asp718I)

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

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

The Acc65I (Asp718I) restriction enzyme recognizes G^GTACC sites and cuts best at 37°C in O buffer (Isoschizomers: Asp718I)
  
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Lambda DNA digested with Acc65I

Lambda DNA digested with Acc65I, 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.

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 EndsBshNI, Bsp1407I, Pfl23II, TatI.
Conditions for 100% Activity
  • 1X Buffer O: 50 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, 100 mM NaCl 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.
NoteAcc65I cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam-, dcm- strain, such as GM2163.
Storage Buffer10 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.

Reaction conditions

Enzyme 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
O (Orange) 37°C 0-20 20-50 100 20-50 20-50 50-100 1X or 2X

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'...GGTACm5CW GG...3'
3'...CCATG GWm5CC...5'		 Impaired
Dcm(CCWGG)5'...Cm5CW GGTACm5CW GG...3'
3'...G GWm5CCATG GWm5CC...5'		 Blocked
CpG5'...GGTACm5C G...3'
3'...CCATG Gm5C...5'		 Impaired
CpG5'...m5C GGTACm5C G...3'
3'... Gm5CCATG Gm5C...5'		 Blocked

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
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
G^GTACC
  • BshNI (BanI)/FastDigest BanI (BshNI)* (G^GTACC)
  • Acc65I (Asp718I)/FastDigest Acc65I
  • BshNI (BanI)/FastDigest BanI (BshNI)
  • BspLI (NlaIV)/FastDigest NlaIV (BspLI)
  • Csp6I (CviQI)/FastDigest Csp6I
  • KpnI/FastDigest KpnI
  • RsaI/FastDigest RsaI
  • Bsp1407I (BsrGI)/FastDigest Bsp1407I (T^GTACA)
  • Pfl23II (BsiWI)/FastDigest BsiWI (Pfl23II) (C^GTACG)
  • TatI/FastDigest TatI* (W^GTACA)
  • TatI/FastDigest TatI* (W^GTACT)
  • Csp6I (CviQI)/FastDigest Csp6I
  • RsaI/FastDigest RsaI

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^GTACCGGTACGTACC
  • [Csp6I (CviQI)/FastDigest Csp6I]
  • Eco105I (SnaBI)/FastDigest SnaBI (Eco105I)
  • MaeII
  • Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)
  • [RsaI/FastDigest RsaI]
  • SetI
  • TaiI (MaeII)/FastDigest TaiI