MspC I (Afl II isoschizomer)

Product Details

MspCI is a restriction enzyme purified from Micrococcus species.

Reagents supplied: 10x R4 and 10x EQ buffer

Unit definition: One unit is the amount of enzyme required to digest 1 μg of Lambda DNA (Hind III digest) in 60 min at 37^oC in a total reaction volume of 50 μl.

Digest Guidelines

• Standard reaction conditions: In order to overcome DNA source variability, a ~10 fold overdigest is suggested:

10U MspC I	1μl
10x EQ buffer	5μl
DNA substrate	1μg
Sterile ultrapure water	Up to 50 μl

• Speed Digest reaction: Incubate for 15 minutes at 37^oC as in standard reaction in 1x R4 buffer (150 mM NaCl, 10 mM Tris-HCl (pH 7.9 @ 25°C), 10 mM MgCl2, 100 μg/ml BSA ) or 1x eQ buffer (20 mM Tris-acetate (pH 7.9 @ 25^oC), 10 mM Mg-acetate, 50 mM K-acetate, 100 μg/ml BSA).

• Activity(%) in EnzyQuest buffers -Double digest tips

R1	R2	R3	R4	EQ
<10	25-50	75-100	100	100

Reactions were performed as for unit definition reaction. The above data could be used as guideline for double digest reactions. Our enzymes are also active and compatible in buffer systems from other vendors.

Heat inactivation: 65^oC for 20 minutes

Methylation Sensitivity:

• dam methylation: Not sensitive
• dcm methylation: Not sensitive
• CpG methylation: Not sensitive

Unit calculation assay conditions:

• 1x R4 buffer
• Lambda DNA (Hind III digest)
• Incubation at 37^oC

Storage conditions:

• 50 mM KCl,
• 10 mM Tris-HCl (pH 7.9 @ 25^oC)
• 0.1 mM EDTA
• 1 mM dithiothreitol
• 200 μg/ml BSA
• 50% glycerol
• Store at -20^oC

Non-specific nuclease activity: 80 units of MspC I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA (Hind III digest) at 37^oC.

Re-ligation and recut: After 10-fold overdigestion with MspC I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.