Restriction endonucleases

Description

Product Details

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 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 Nhe 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 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
100	50-75	0-20	<10	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: Blocked by some combinations of overlapping

Unit calculation assay conditions:

• 1x R5 buffer
• Lambda DNA (Hind III digest)
• Incubation at 37^oC

Storage conditions:

• 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
• 50% glycerol
• Store at -20^oC

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 37^oC.

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.