|Source||Paracoccus carotinifaciens 3K|
|Substrate specificity||The enzyme cleaves only C5-methylated DNA and does not cut unmodified DNA .|
The enzyme activity depends on nucleotides neighboring to a recognition site and a number of methylated cytosines.
Optimal recognition site (100% activity ):
|Assayed on||pMHgaI/DriI is a linearized plasmid pMHgaI, which carries genes of DNA-methyltransferases M1.HgaI (recognition sequence 5`-GCGTC-3`) and M2.HgaI (5`-GACGC-3`) and includes a unique PcsI canonical site:|
|Unit definition||One unit is defined as the amount of enzyme required to digest a unique site|
in 1 μg of DNA pMHgaI/DriI in 1 hour at 37°C in a total reaction volume of 50 μl.
|PcsI activity assay on DNA pMHgaI/DriI |
1 and 6 – 1 Kb SE DNA-markers
2 – Control pMHgaI/DriI
3 – 0.5 μl Pcs I
4 – 1 μl Pcs I
5 – 2 μl Pcs I
Products were separated in 1,4% agarose gel in Buffer TAE.
|Optimal SE-buffer||SE-buffer PcsI (10 mM Tris-HCl (pH 8.3 at 25°C); 20mM NaCl, 3 mM MgCl2, 1 mM DTT.)|
|Enzyme activity (%)|
|50 - 75||25 - 50||0 - 10||10 - 25||50 - 75||20|
|Reaction buffer||SE-buffer PcsI,(10 mM Tris-HCl (pH 8.3 at 25°C); 20mM NaCl, 3 mM MgCl2, 1 mM DTT.)|
|Storage conditions||10 mM Tris-HCl (pH 7.5); 200 mM NaCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 0.1 mg/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 37°C in a total reaction volume of 50 μl.
|Reagents Supplied with Enzyme||
10 X SE-buffer PcsI|
|Methylation sensitivity||The enzyme cleaves only C5-methylated DNA and does not cut unmodified DNA .
|Inactivation 20 minutes under||65oC|
|Notes||When using a buffer other than the optimal (suppied) SEBuffer, it may be necessary to add more enzyme to achieve complete digestion.|
|MSDS:||Download MSDS as PDF|
|References:||1. Chernukhin V.A., Nayakshina T.N., Tarasova M.V., Golikova L.N., Akishev A.G., Dedkov V.S., Mikhnenkova N.A., Degtyarev S.Kh. Bacterial strain Paracoccus carotinifaciens 3K- producer of PcsI site specific endonuclease. // Russian Federation patent RU 2377294 C1 (2009). |