NheI

Blue Ice

Molecular Biology Grade

5'-G^C T A G C-3'
3'-C G A T C^G-5'

Source:
Neisseria mucosa heidelbergensis

Unit Definition:
One unit is defined as the amount of enzyme required to digest 1ug of lambda DNA-HindIII fragments in 1 hour at 37°C in 50ul of assay buffer.

Concentration:
10 units/ul

Supplied With:
R1625: Restriction Enzyme Buffer A, 10X: Supplied as a liquid in 33mM Tris-acetate, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA (pH 7.9 at 37°C) when diluted to 1X.

Diluent Buffer:
10mM Tris-HCl, (pH 7.4 at 25°C), 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol.

Storage Buffer:
Supplied as a liquid in 10mM Tris-HCl, pH 8.0 at 25ºC, 50mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA, 50% glycerol.

Incubation Temp:
37°C

Compatible Ends:
BcuI, Eco130I, XbaI, XmaJI

Number of Recognition Sites in DNA Molecules:
Lambda: 1PhiX174: 0
M13mp18/19:0pBR322: 1
pUC18/19: 0pUC57: 0
pTZ19R/U: 0

*Note:
Supercoiled plasmids may require up to 10-fold more Nhel for complete digestion than linear DNAs (e.g. 10units are required to cleave 1ug of pBR322 DNA).

Thermal Inactivation:
NheI is inactivated by incubation at 65°C for 20min.

Stability During Prolonged Incubation:
≥ 0.2units of NheI is required for complete digestion of 1ug of lambda DNA in 16 hours at 37ºC.

Enzyme Properties:
Methylation Effects on Digestion:
Dam, Dcm, EcoKl, EcoBl: never overlaps - no effect
CpG: May overlap - cleavage impaired

Digestion of Agarose-embedded DNA:
A minimum of 5 units of Nhel is required for complete digestion of 1ug of agarose-embedded lambda DNA in 16hrs

Overdigestion Assay:
No detectable change in the specific fragmentation pattern is observed after 80-fold overdigestion (5u/ug lambda DNA x 16 hours) with Nhel.

Ligation Recutting Assay:
The ligation and recleavage assay was replaced with L0 assay after validating experiments showed L0 assay ability to trace nuclease and phosphatase activities with sensitivity that is higher than L/R by a factor of 100.

Labeled Oligonucleotide (LO) Assay:
No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10units of NheI for 4 hours.

Storage and Stability:
For long-term storage, store at -20°C. Aliquots are stable for 6 months after receipt at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.