NmuCI (Tsp45I)

Blue Ice

Molecular Biology Grade

C 5'-^G T G A C-3'
3'-C A C T G^-5' G

Concentration:
10u/ul

Buffer:
10mM Tris-HCl (pH 8.5), 10mM MgCl2, 100mM KCl and 0.1mg/ml BSA. Incubate at 37°C.

Diluent Buffer:
10mM Tris-HCl (pH 7.5 at 25°C), 100mM KCl, 0.1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol. For longer periods–the Storage Buffer should be used.

Storage Buffer:
10mM Tris-HCl (pH 7.4 at 25°C), 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA and 50% glycerol.

Overdigestion Assay:
No detectable change in the specific fragmentation pattern is observed after 320-fold overdigestion (20u/ug lambda DNA x 16 hours) with NmuCI.

Ligation/Recutting Assay:
After 50-fold overdigestion (3u/ug DNA x 17 hours) with NmuCI, more than 95% of the DNA fragments can be ligated at a 5'-termini concentration of 0.8uM. More than 95% of these can be recut.

Labeled Oligonucleotide (LO) Assay:
No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10 units of restriction endonuclease for 4 hours.

Methylation Effects:
GT(C/G)m5C is cleaved by NmuCI at a significantly slower rate. Overlapping CG methylation may influence DNA cleavage.

Stability during Prolonged Incubation:
A minimum of 0.1 units of enzyme is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C.

Thermal Inactivation:
Enzyme is inactivated by incubation at 65°C for 20min.

Compatible Ends:
Eco91I (G^GTCACC) MaeIII (^GTGAC).

Number of Recognition Sites in DNA:

Lambda: 81
PhiX174: 8
M13mp18/19: 8
pBR322: 9
pUC18/19: 4
pUC57: 4
pTZ19R/U: 4
pBluescriptIIKS(-/+): 4
pBluescriptIISK(-/+): 4
pACYC177: 5
pACYC184: 7

Unit Definition:
One unit is defined as the amount of enzyme required to digest 1ug of lambda DNA in 1 hour at 37°C in 50ul of assay buffer.