Recognition Sequence :
AGC↑GCT
TCG↓CGA
Unit Definition :
One unit of the enzyme is the amount required to hydrolyze 1 μg of Lambda DNA (BamHI-digest) in 1 hour at 37°C in a total reaction volume of 50 μl.
Reaction Temperature :
37°C
Storage Buffer :
10 mM Tris-HCl (pH 7.6); 50 mM NaCl; 0.1 mM EDTA; 1 mM DTT; 200 μg/ml BSA; 50% glycerol
Ligation :
After 10-fold overdigestion with enzyme more than 80% of DNA pBR322 fragments can be ligated and recut.
Source :
An E.coli strain that carries the cloned Afe I gene from Alcaligenes faecalis T2774
Assayed on :
Lambda DNA (BamHI-digest)
Working buffer :
Y (33 mM Tris-acetate (pH 7.9 at 25°C); 10 mM magnesium acetate; 66 mM potassium acetate; 1 mM DTT.)
Non-specific hydrolisis :
No nonspecific activity was detected after incubation of 1 μg of DNA with 40 u.a. of enzyme for 16 hours at 37°C.
Concentration, u.a./ml :
10000
Inactivation :
20min Under 65°C
Reagents Supplied :
10 X SE-buffer B, BSA
Notes :
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.
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