Bis I

Supplied in: 10 mM Tris-HCl (pH 7.5); 100mM NaCl; 0.1mM EDTA; 7mM 2-mercaptoethanol; 50% glycerol; Store at -20°C

Reaction Conditions:

1X SEBuffer Bis I

Incubate at  37°C

Warranty period for the enzyme storage at -20˚C is two years from the date of the last assay indicated on the enzyme vial.

1X SEBuffer BisI (pH 9.0 @ 25ºC)

10 mM Tris-HCl, 150 mM KCl, 10 mM MgCl2, 1 mM DTT

Unit Definition: One unit is defined as the amount of enzyme required to cleave 1 pmol of the double-stranded oligonucleotide with the following structure

5′  GCTTGTACTTTAG(5mC)GGCATTGATTCTCACCACG  3′

3′ CGAACATGAAATCGC(5mC)GTAACTAAGAGTGGTGC  5′

in 1 hour at 37°C in a total reaction volume of 20μl.

Reagents supplied with enzyme: 10X SEBuffer Bis I

Heat Inactivation: Yes   (65°C for 20 minutes)

Quality Control Assays

16-Hour Incubation: No nonspecific activity was detected after incubation of 1 μg of pFsp4HI1  DNA (BamHI digest) with 1 unit of BisI for 16 hours at 37°C. The pFsp4HI1 plasmid carries a gene for Fsp4HI DNA-methyltransferase, which modifies DNA forming 5′-G(5mC)NGC-3’/3′-CGN(5mC)G-5′.

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          10-25%

SEBuffer G          25-50%

SEBuffer O          50-75%

SEBuffer W         75-100%

SEBuffer Y          50-75%

SEBuffer ROSE  30%

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