Nhe I
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Nhe I Technical BrochureTechnical Brochure
NheI is a restriction enzyme purified from Neisseria mucosa heildelbergensis (ATCC 25999).

 

Unit substrate: Lambda DNA (HindIII digest).

 

Unit calculation assay conditions: 50 mM potassium acetate, 20 mM Tris-acetate (pH 7.9 @ 25°C), 10 mM magnesium acetate, 1 mM dithiothreitol, 100 μg/ml BSA. Incubate at 37oC.

 

Absence of contaminants: 80 units of NheI do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of λ DNA/Hind III digest at 37oC. After 100-fold overdigestion with NheI, greater than 98% of the DNA fragments can be ligated and recut.

 

Storage buffer: 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 and 50% glycerol. Store at -20oC.

 

Heat inactivation: 65°C for 20 minutes..

 

Methylation Sensitivity:

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: Blocked by some combinations of overlapping

 

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.

Star activity: Low salt, high glycerol (>5%) concentrations, pH >8.0 or large excess of the enzyme may result in star activity.   

 

Percent Activity in MINOTECH Buffers
 L SH 
 100 50-75  0-20  <10  100  100 
 
 
General reaction mixture:
10U NheI 1μl
10x A or K buffer*  2μl 
DNA substrate  <1μg
Sterile ultrapure water  Up to 20 μl
Incubate for 15 min at 37oC  
*In the case of A buffer we recommend the addition of BSA to a final concentration of 100 μg/ml. 

 

Reagents supplied: 10x A and 10x K buffer