|Source||Kocurea rosea 307|
|Substrate specificity||The enzyme cleaves C5-methylated DNA and doesn't cut unmodified DNA . |
Kro I doesn`t cleave DNA modified with MspI DNA-methyltransferase
|Assayed on||DNA pMHpaII 1/DriI is a linearized plasmid pMHpaII 1.
pMHpaII 1 carries a gene of DNA-methyltransferase M.HpaII, which methylates sites 5`-CCGG-3` producing 5`-C(5mC)GG-3`/3`-GG(5mC)C-5`, and includes three canonical sites |
|Unit definition||One unit is defined as the amount of enzyme required to hydrolyze completely 1 μg of linearized plasmid pMHpaII 1 in 1 hour at 37°C in a total reaction volume of 50 μl.
|KroI activity assay on DNA pMHpaII 1/DriI |
1 and 6 – 1 Kb SE DNA-markers.
2 – Control DNA pMHpaII 1/DriI,
3 – 0.5 μl Kro I
4 – 1 μl Kro I
5 – 2 μl Kro I
Products were separated in 1,4% agarose gel in Buffer TAE.
|Optimal SE-buffer||G (10 mM Tris-HCl (pH 7.6 at 25°C); 10 mM MgCl2; 50 mM NaCl; 1 mM DTT.)|
|Enzyme activity (%)|
|50 - 75||100||25 - 50||50 - 75||75 - 100||100|
|Reaction buffer||SE-buffer G, (10 mM Tris-HCl (pH 7.6 at 25°C); 10 mM MgCl2; 50 mM NaCl; 1 mM DTT.)|
|Storage conditions||10 mM Tris-HCl (pH 7.5); 200 mM NaCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 200 μg /ml BSA; 50% glycerol; Store at -20°C.|
|Non-specific hydrolisis||No detectable degradation of 1μg of Lambda DNA was observed after incubation with 1 units of enzyme for 16 hours at 30°C in a total reaction volume of 50 μl.
|Reagents Supplied with Enzyme||
10 X SE-buffer G|
|Methylation sensitivity||The enzyme cleaves C5-methylated DNA and doesn't cut unmodified DNA .|
|Inactivation 20 minutes under||65oC|
|MSDS:||Download MSDS as PDF|
|References:||1. V.A. Chernukhin, E.V. Kileva, J.E. Tomilova, A.A. Boltengagen, V.S. Dedkov, N.A. Mikhnenkova, D.A. Gonchar, L.N. Golikova, S.Kh. Degtyarev A new methyl-directed site-specific endonuclease KroI recognizes and cuts DNA sequence 5’-G^C(5mC)GGC-3’ // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.7, No 1, pp 14-20, 2011
2. Chernukhin V.A., Zhuravleva R.O., Tarasova G.V., Boltengagen A. A., Akishev A.G., Mikhnenkova N.A., Degtyarev S.Kh. Bacterial strain Kocuria rosea - producer of KroI site specific endonuclease. // Russian Federation patent RU 2394099 C1 (2010).