Thermo Scientific™ AarI (2 U/μL)

Description

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.

5'...C A C C T G C (N)₄▵...3'

3'...G T G G A C G (N)₈▵...5'

Conditions for 100% Activity:

• [1X Buffer AarI] + oligonucleotide:
• [10mM Bis-Tris Propane-HCl (pH 6.5 at 37°C), 10mM MgCl₂, 100mM KCl, 0.1mg/mL BSA] + 0.5μM of oligonucleotide (see Note)
• Incubate at 37°C

Storage Buffer:

• AarI is supplied in:
• 10mM potassium phosphate (pH 7.4 at 25°C), 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/mL BSA and 50% (v/v) glycerol

Ligation and Recleavage:

• After 10-fold overdigestion with AarI, more than 95% of the DNA fragments can be ligated and recut

Methylation Effects:

• Dam: never overlaps — no effect
• Dcm: never overlaps — no effect
• CpG: may overlap — cleavage impaired
• EcoKI: never overlaps — no effect
• EcoBI: may overlap — effect not determined

Digestion of Agarose-embedded DNA:

• Minimum 5 units of the enzyme are required for digestion of 1μg of agarose-embedded lambda DNA in 16 hours

Note:

For cleavage with AarI at least two copies of its recognition sequence are required. Inclusion of 0.5μM oligonucleotide with the AarI recognition sequence in the reaction mixture significantly improves cleavage of DNAs, especially of those with a single AarI site. Still, a complete cleavage of some substrates with AarI is difficult to achieve. Greater than 10-fold overdigestion with AarI may result in star activity. AarI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.