Product Catalog

Sequence specificity
Aat II recognizes the sequence GA*CGT↓*C and generates fragments with 3´-cohesive termini.

Source
Acetobacter aceti

Compatible ends
Aat II  ends are not compatible with ends generated by any other known restriction enzyme.

Isoschizomer
Aat II is an isoschizomer of Ssp 5230 I and Zra I.

Methylation sensitivity
Aat II is inhibited by the presence of 5-methylcytosine at either C residue, as indicated (*) on the recognition sequence.

Activity in SuRE/Cut buffer System
Buffer printed in bold face type is the buffer recommended for optimal activity:

Activity in PCR buffer
Not tested

Incubation temperature
37°C

Unit definition
One unit is the enzyme activity that completely cleaves 1 μg λDNA in 1 h at 37°C in a total volume of 25 μl SuRE/Cut buffer A.

Heat inactivation
The enzyme can be heat inactivated by incubating it for 15 min at 65°C.

Number of cleavage sites on different DNAs

PFGE tested
Aat II has been tested in Pulsed-Field Gel Electrophoresis (on bacterial chromosomes). For cleavage of genomic DNA (E. coli C 600) embedded in agarose for PFGE analysis, we recommend using 10 units of enzyme/μg DNA and 4 h incubation time.

1. Aat II
2. SuRE/Cut buffer A (10x)

Absence of nonspecific endonuclease activities
1 μg  λDNA is incubated for 16 h in 50 μl SuRE/Cut buffer A with excess of Aat II. The number of enzyme units that may be included in the incubation mixture without changing the enzyme-specific pattern is stated under "Endo" (on the product label).

Absence of 5´-exonuclease/ 5´-phosphatase activities
5´-[³²P] terminally labeled λ × Hpa II fragments are incubated with Aat II for 4 h at 37°C in SuRE/Cut buffer A.  After separation by TLC, the amount of radioactivity released is calculated as a percentage of input radioactivity, then divided by the number of units of enzyme. That number is stated under “5´-Exo” (on the product label).

Absence of 3´-exonuclease activity
3´-[³²P] terminally labeled λ × Hpa II fragments  are incubated with Aat II for 4 h at 37°C in SuRE/Cut buffer A.  After separation by TLC,  the amount of radioactivity released is calculated as a percentage of input radioactivity, then divided by the number of units of enzyme. That number is stated under  “3´-Exo” (on the product label).

Ligation and recutting assay
Aat II fragments obtained by complete digestion of 1 μg pBR322 DNA are ligated for 16 h at 4°C with 0.1 U T4-DNA ligase in 10 μl buffer that contains 66 mM Tris-HCl, 5 mM MgCl₂, 1 mM dithioerythritol, 1 mM ATP, pH 7.5 (at 20°C).
The percentages of product that can be ligated and subsequently recut with Aat II  (yielding the typical pattern of pBR322 × Aat II fragments) are stated under "Lig" and "Rec" (on the product label).

• Rapid DNA Ligation Kit;
  For rapid and efficient DNA ligation.
• Klenow Enzyme Labeling grade, from Escherichia coli lysogenic NM 964;
  
• DNA Molecular Weight Marker XIV (100 bp ladder);