NheI is a restriction enzyme purified from Neisseria mucosa heildelbergensis (ATCC 25999).
Reagents supplied: 10x R5 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 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 Nhe 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 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: Not sensitive
dcm methylation: Not sensitive
CpG methylation: Blocked by some combinations of overlapping
Unit calculation assay conditions:
1x R5 buffer
Lambda DNA (Hind III digest)
Incubation at 37oC
200 mM NaCl,
10 mM Tris-HCl (pH 7.5)
0.1 mM EDTA
1 mM dithiothreitol
0.15% Triton X-100
200 μg/ml BSA
Store at -20oC
Non-specific nuclease activity: 80 units of Nhe I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA (Hind III digest) at 37oC.
Re-ligation and recut: After 100-fold overdigestion with Nhe I, greater than 98% of the DNA fragments can be ligated and recut.
Star activity: Low salt, high glycerol (>5%) concentrations, pH >8.0 or large excess of the enzyme may result in star activity.
Note: Activity inhibited by salt concentrations >100mM. Cleaves to leave a 5’ CTAG extension which can be efficiently ligated to DNA fragments generated by AvrII, SpeI, or XbaI.
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