Source: An E.coli strain that carries the cloned Pcs I gene from Paracoccus carotinifaciens 3K.
One unit is defined as the amount of enzyme required to digest 1 µg of pMHpySE49/DriI DNA in 1 hour at 37°C in a total reaction volume of 50 µl. As a result, a linearized plasmid pMHpySE49/DriI disappears, and two DNA fragments are produced.
|PcsI activity assay on pMHpySE49/DriI DNA
Lanes:1 and 7 – 1 Kb SE DNA-ladders
2 – Control pMHpySE49/DriI
3 – 0.25 μl Pcs I
4 – 0.5 μl Pcs I
5 – 1 μl Pcs I
6 – 2 μl Pcs I
Products were separated in 1,4% TAE agarose gel.
pMHpySE49 plasmid DNA, linearized with DriI (pMHpySE49/DriI).
pMHpySE49 (3536 bp) was obtained by a ligation of the vector pMTL22/FauNDI/SmaI and a PCR-fragment containing M.HpySE526I (forms 5′-A(5mC)GT-3′) gene and
Thus, the plasmid has the optimal site of PcsI:
Optimal SE-buffer: SE-buffer PcsI
Enzyme activity (%):
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 .
Notes: When using a buffer other than the optimal (suppied) SEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
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).