Source: Alcaligenes faecalis T2774.
5000-10000 u/ml Store at -20°C
Source: Alcaligenes faecalis T2774
Supplied in: 10 mM Tris-HCl (pH 7.6); 50 mM NaCl; 0,1 mM EDTA; 200 µg/ml BSA; 1mM DTT; 50% glycerol.
Reaction Conditions: 1X SEBuffer Y
Incubate at 37°C.
1X SEBuffer Y
33 mM Tris-Ac (pH 7.9 @ 25ºC), 66 mM KAc, 10 mM MgAc, 1 mM DTT
Unit Definition: One unit is defined as the amount of enzyme required to digest 1µg of l DNA (BamH I-digest ) in 1 hour at 37 ºC in a total reaction volume of 50 µl.
Quality Control Assays
Ligation: After 10-fold overdigestion with Afe I, approximately 80% of DNA pBR322 fragments can be ligated with T4 DNA Ligase and recut.
16-Hour Incubation: A 50 µl reaction containing 1µg of DNA and 10 units of enzyme incubated for 16 hours resulted in the same pattern of DNA band as a reaction incubated for 1 hour.
Oligonucleotide Assay: No detectable degradation of a single- and double-stranded oligonucleotide wasobserved after incubation with 10 units of enzyme for 3 hours.
Activity in SEBuffers:
SEBuffer B 10-25%
SEBuffer G 25-50%
SEBuffer O 75-100%
SEBuffer W 75-100%
SEBuffer Y 100%
SEBuffer ROSE 100%
When using a buffer other than the optimal (suppied) SEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Heat Inactivation: Yes (65°C for 20 minutes)
Reagents supplied with enzyme: 10X SEBuffer Y.
Note: The minimum number of units that resulted in complete digestion of 1 μg of substrate DNA in 16 hours is 0,25. AfeI cleaves supercoiled and linear plasmid DNA (pBR322) at a roughly equal rate. AfeI cleaves Lambda DNA/BamHI digest at a rate 3-4 times higher than plasmid DNA.