Fat I

Enzyme nameFat I
PrototypeNlaIII
Known analogsCviAII, Fae I, Hin1II, Hsp92II, NlaIII
SKUSE-E155
Turbo versionNot available
High-concentration versionNot available
Restriction typeType IIPiType IIP enzymes are classical Type II restriction enzymes with palindromic recognition sequences and symmetric cleavage sites.
Recognition site
5'… ▼CATG …3'
3'… GTAC▲ …5'
SourceAn E.coli strain that carries the cloned Fat I gene from Flavobacterium aquatile NL3
Optimal bufferSE-buffer G (10 mM Tris-HCl (pH 7.6 at 25°C); 10 mM MgCl2; 50 mM NaCl; 1 mM DTT.)
Optimal temperature55 °C
Inactivation temperature65 °C
Buffer activity
BGOWYROSE
10100251050100
Unit definitionOne unit of the enzyme is the amount required to hydrolyze 1 μg of pUC19 DNA in 1 hour at 55°C in a total reaction volume of 50 μl.
Assayed onpUC19 DNA
Storage conditions10 mM Tris-HCl (pH 7.5); 50 mM KCl; 0,1 mM EDTA; 1 mM DTT; 200 μg/ml BSA; and 50% glycerol. Store at -20°C.
LigationAfter 2-fold overdigestion with enzyme more than 90% of the DNA fragments can be ligated and recut.
Nonspecific hydrolysisNo nonspecific activity was detected after incubation of 1 μg of DNA with 3 u.a. of enzyme for 16 hours at 55°C.
Methylation sensitivityBlocked by m CATG methylation
Reagents supplied with enzyme10 X SE-buffer G
NotesThe minimum number of units that resulted in complete digestion of 1 μg of substrate DNA in 16 hours is 0,25. FatI cleaves linear plasmid DNA at a rate 1.5-2 times higher than supercoiled plasmid DNA.
References
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