Product info: Pcs I


Name
Pcs I
Cat. #E505E506
Package, u.a.50250
Concentration, u.a./ml10001000

Recognition site
(5mC)GNNNNN↑NN(5mC)G
G(5mC)NN↓NNNNNG(5mC)
SourceParacoccus carotinifaciens 3K
Substrate specificityThe enzyme cleaves only C5-methylated DNA and does not cut unmodified DNA [1].

The enzyme activity depends on nucleotides neighboring to a recognition site and a number of methylated cytosines.

Optimal recognition site (100% activity ):
5'-W(5mC)GNNNNNNN(5mC)GW-3'/ 3'-WG(5mC)NNNNNNNG(5mC)W-5`
Assayed onpMHgaI/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:
5`-W(5mC)GNNNNNNN(5mC)GW-3`/
3`-WG(5mC)NNNNNNNG(5mC)W-5` [1].
Unit definitionOne unit is defined as the amount of enzyme required to digest a unique site
5`-A(5mC)GNNNNNNN(5mC)GT-3`
in 1 μg of DNA pMHgaI/DriI in 1 hour at 37C in a total reaction volume of 50 μl.
PcsI activity assay on DNA pMHgaI/DriI

Lanes:
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.

PcsI activity assay on DNA pMHgaI/DriI
Optimal SE-bufferSE-buffer PcsI (10 mM Tris-HCl (pH 8.3 at 25°C); 20mM NaCl, 3 mM MgCl2, 1 mM DTT.)
Enzyme activity (%)
BGOWYRose
50 - 7525 - 500 - 1010 - 2550 - 7520
Reaction bufferSE-buffer PcsI, (10 mM Tris-HCl (pH 8.3 at 25°C); 20mM NaCl, 3 mM MgCl2, 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; 0.1 mg/ml BSA, 50% glycerol; Store at -20C.
Non-specific hydrolisisNo detectable degradation of 1μg of Lambda DNA was observed after incubation with 1 units of enzyme for 16 hours at 37C in a total reaction volume of 50 μl.
Reagents Supplied with Enzyme 10 X SE-buffer PcsI
Methylation sensitivityThe enzyme cleaves only C5-methylated DNA and does not cut unmodified DNA [1].
Inactivation 20 minutes under
65oC
NotesWhen 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).