Restriction endonucleases

Description

Product Details

NruI is a restriction enzyme purified from Nocardia rubra.

Reagents supplied: 10x Nru I and 10x EQ buffer

Unit definition: One unit is the amount of enzyme required to digest 1 μg of Lambda DNA 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 Nru 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 NruI buffer (100 mM KCl, 50 mM Tris-HCl (pH 8.0 @ 25^oC), 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	<10	75-100	50-75	75-100

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: Blocked by overlapping
• dcm methylation: Not sensitive
• CpG methylation: Blocked

Unit calculation assay conditions:

• 1x NruI buffer
• Lambda DNA
• Incubation at 37^oC

Storage conditions:

• 50 mM KCl,
• 10 mM Tris-HCl (pH 7.4)
• 0.1 mM EDTA
• 1 mM dithiothreitol
• 200 μg/ml BSA
• 50% glycerol
• Store at -20^oC

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

Re-ligation and recut: After 10-fold overdigestion with Nru I, less than 20% of the DNA fragments can be ligated.

Star activity: Large excess of the enzyme results in the appearance of star activity. After 2 hours of a 10-fold overdigestion did not result in star activity of Nru I in either eQ or unique buffer (Nru I 10 X).