Pcs I

Supplied in:

10 mM Tris-HCl (pH 7.5); 200 mM NaCl; 0,1 mM EDTA; 100 µg/ml BSA; 7 mM  2-mercaptoethanol; 50% glycerol.

Reaction Conditions:

1X SEBuffer PcsI

Incubate at  37°C.

1X SEBuffer PcsI (pH 8.3 @ 25ºC)

10 mM Tris-HCl, 20 mM NaCl, 3 mM MgCl2, 1 mM DTT

Unit Definition:

One 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. DNA 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(5mC)GNNNNNNN(5mC)GW-3'/3'-WG(5 m C)NNNNNNNG(5mC)W-5'. The enzyme activity depends on number and position of methylated nucleotides in the recognition sequence.

Optimal recognition site (100% activity ):

5'-W(5mC)GNNNNNNN(5mC)GW-3'/ 3'-WG(5mC)NNNNNNNG(5mC)W-5`

Quality Control Assays

16-Hour Incubation:

No detectable degradation of 1 µg of λ DNA was observed after incubation with 1 units of enzyme for 16 hours at 37ºC

Oligonucleotide Assay:

No detectable degradation of a single- and double-stranded oligonucleotide was observed after incubation with 1 units of enzyme for 3 hours.

Enzyme Properties

Activity in SEBuffers:

SEBuffer B        50-75%

SEBuffer G        25-50%

SEBuffer O                0%

SEBuffer W        10-25%

SEBuffer Y          50-75%

SEBuffer ROSE       20% 

When using a buffer other than the optimal (suppied) SEBuffer, it may be necessary to add more enzyme to achieve complete digestion.

Reagents supplied with enzyme:

10X SEBuffer PcsI

Heat Inactivation: 

Yes   (65°C for 20 minutes)