| Recognition site | W(5mC)GNNNN↑NNN(5mC)GW
WG(5mC)NNN↓NNNNG(5mC)W |
| Source | Paracoccus carotinifaciens 3K |
| Unit | One unit is defined as the amount of enzyme required to digest a unique site
5`-A(5 m C)GNNNN^NNN(5 m C)GT-3`
in 1 μg of DNA pMHgaI/DriI in 1 hour at 37°C in a total reaction volume of 50 μl. |
| 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: | | B | G | O | W | Y | | 50 - 75 | 25 - 50 | 0 - 10 | 10 - 25 | 50 - 75 |
|
| Optimal temperature | 37oC |
| Assayed on | pMHgaI/DriI is a linearized plasmid pMHgaI, which included a genes of DNA-methyltransferases M1.HgaI (recognition sequence 5`-GCGTC-3`) and M2.HgaI (5`-GACGC-3`) and contains a unique PcsI canonical site:
5`-W(5 m C)GNNNN^NNN(5 m C)GW-3`/
3`-WG(5 m C)NNN^NNNNG(5mC)W-5` |
| 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.
No detectable degradation of a single- and double-stranded oligonucleotide
was observed after incubation with 0,5 unit of enzyme for 3 hours at 37°C.
|
| 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.
Reaction conditions:
1 x SE-buffer PcsI, 10 % DMSO |
