Afe I

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 was

observed after incubation with 10 units of enzyme for 3 hours.

Enzyme Properties

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.