Product info: Pcs I


Name
Pcs I
Cat. #E505E506
Package, u.a.2001000
Concentration, u.a./ml50005000

Recognition site
(5mC)GNNNNN↑NN(5mC)G
G(5mC)NN↓NNNNNG(5mC)
SourceAnE.coli strain that carries the cloned P cs I gene fromParacoccus carotinifaciens3K
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 onpMHpySE49 plasmid DNA, linearized with Dr iI (pMHpySE49/DriI). pMHpySE49 (3536 bp) was obtained by a ligation of the vector pMTL22/FauNDI/SmaI and a PCR-fragment containing M.HpySE526I (f orms 5'-A(5mC)GT-3') gene and DNA sequence:
5'-GACGTAT AAAACGTT-3'
3'-CTGCAT ATTTTGCAA-5'
Thus, the plasmid has the optimal site of PcsI:
5'-A(5mC)GNNNNNNN(5mC)GT-3'
3'-TG(5mC)NNNNNNNG(5mC)A-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 37°C in a total reaction volume of 50 μl.
PcsI activity assay on DNA pMHpySE49/DriI

Lanes:
1 and 7 1 Kb SE DNA-markers
2 Control pMHpySE49/DriI
3 0.5 μl Pcs I (diluted 1/10)
4 1 μl Pcs I (diluted 1/10)
5 2 μl Pcs I (diluted 1/10)
6 1 μl Pcs I concentrated

Products were separated in 1,0% 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 - 7550 - 750 - 1010 - 2550 - 7550
Reaction bufferSE-buffer PcsI
Optimal temperature
37oC
Storage conditions15 mM Tris-HCl (pH 7.5); 250 mM NaCl; 0,1 mM EDTA; 0,05% Triton X-100, 7 mM 2-mercaptoethanol; 100 g/ml BSA; 50% glycerol. Store at -20°C.
Non-specific hydrolisisNo detectable degradation of 1μg of Lambda DNA was observed after incubation with 10 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 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).