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 37oC in a total reaction volume of 50 μl.
Standard reaction conditions: In order to overcome DNA source variability, a ~10 fold overdigest is suggested:
10U Nru I
10x EQ buffer
Sterile ultrapure water
Up to 50 μl
Speed Digest reaction: Incubate for 15 minutes at 37oC as in standard reaction in 1x NruI buffer (100 mM KCl, 50 mM Tris-HCl (pH 8.0 @ 25oC), 10 mM MgCl2, 100 μg/ml BSA) or 1x EQ buffer (20 mM Tris-acetate (pH 7.9 @ 25oC), 10 mM Mg-acetate, 50 mM K-acetate, 100 μg/ml BSA).
Activity(%) in EnzyQuest buffers-Double digest tips
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: 65oC for 20 minutes
dam methylation: Blocked by overlapping
dcm methylation: Not sensitive
CpG methylation: Blocked
Unit calculation assay conditions:
1x NruI buffer
Incubation at 37oC
50 mM KCl,
10 mM Tris-HCl (pH 7.4)
0.1 mM EDTA
1 mM dithiothreitol
200 μg/ml BSA
Store at -20oC
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 37oC.
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).
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