1000 u/ml store at -20°C
Source: Planomicrobium koreense 78k
The enzyme cleaves C5-methylated DNA and doesn't cut unmodified DNA.
PkrI cleaves DNA sequence 5`- GCNGC-3`/3`-CGNCG-5', if at least three 5-methylcytosines are present in the recognition site (N isn`t considering) .
Optimal recognition sites (100% activity):
20 mM Tris-HCl (pH 7.4); 200 mM NaCl; 0,1 mM EDTA; 7 mM 2-mercaptoethanol; 50% glycerol.
1X SEBuffer Y.
Incubate at 37°C.
1X SEBuffer Y (pH 7.9 @ 25ºC)
33 mM Tris-Ac, 66 mM KAc, 10 mM MgAc, 1 mM DTT
One unit is defined as the amount of enzyme required to hydrolyze at least one of three canonical sites: 5`-G(5mC)NG(5mC)-3`/3`-CGN(5mC)G-5` in 1 μg of linearized plasmid pFsp4HI3 in 1 hour at 37°C in a total reaction volume of 50 μl. DNA pFsp4HI3/DriI is a linearized plasmid pFsp4HI3, which carries a gene of DNA-methyltransferase M.Fsp4HI and includes three canonical sites: 5`-G(5mC)NG(5mC)-3`/3`-CGN(5mC)G-5` .
Quality Control Assays
No detectable degradation of 1μg of Lambda DNA was observed after incubation with 2 units of enzyme for 16 hours at 37°C in a total reaction volume of 50 μl.
No detectable degradation of a single- and double-stranded oligonucleotide was observed after incubation with 2 units of enzyme for 3 hours.
Activity in SEBuffers:
SEBuffer B 50-75%
SEBuffer G 75-100%
SEBuffer O 10-25%
SEBuffer W 25-50%
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 a complete digestion.
Reagents supplied with enzyme:
10X SEBuffer Y
Yes (65°C for 20 minutes)