Product info: Kro I


Name
Kro I
Cat. #E541E542
Package, u.a.50250
Concentration, u.a./ml10001000

Recognition site
G↓C(5mC)GGC
CGG(5mC)C↑G
SourceKocurea rosea 307
Substrate specificityThe enzyme cleaves C5-methylated DNA and doesn't cut unmodified DNA [1].
Kro I doesn`t cleave DNA modified with MspI DNA-methyltransferase

Recognition site
G↓C(5mC)GGC / CGG(5mC)C↑G.
Assayed onDNA 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
5`-GC(5mC)GGC-3`/3`-CGG(5mC)CG-5`.
Unit definitionOne 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

Lanes:
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.
KroI activity assay on DNA pMHpaII 1/DriI
Optimal SE-bufferG (10 mM Tris-HCl (pH 7.6 at 25°C); 10 mM MgCl2; 50 mM NaCl; 1 mM DTT.)
Enzyme activity (%)
BGOWYRose
50 - 7510025 - 5050 - 7575 - 100100
Reaction bufferSE-buffer G, (10 mM Tris-HCl (pH 7.6 at 25°C); 10 mM MgCl2; 50 mM NaCl; 1 mM DTT.)
Optimal temperature
37oC
Storage conditions10 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 hydrolisisNo 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 sensitivityThe enzyme cleaves C5-methylated DNA and doesn't cut unmodified DNA [1].
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).