Sourse:An E.colistrain that
carries the cloned Afe I gene from Alcaligenes faecalis T2774.
10 mMTris-HCl (pH 7.6); 50 mM NaCl;
0,1 mMEDTA;200µg/ml BSA;1mM DTT;
Incubate at 37°C.
33 mMTris-Ac (pH 7.9 @ 25ºC) 66 mM KAc
10 mMMgAc 1 mM DTT
One unit is defined as the amount
of enzyme required to digest 1µg oflDNA (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 andrecut.
A 50µl reaction containing 1µg
10 units of enzyme incubated for 16 hours resulted
in the same pattern of DNA band as a reaction
incubated for 1 hour.
No detectable degradation of a single-
and double-stranded oligonucleotide was
observed after incubation with 10 units of
enzyme for 3 hours.
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
Yes(65°C for 20
Reagents Supplied with
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.