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  Restriction endonucleases

Not I

NotI is a restriction enzyme purified from Nocardia otitidis-caviarum.

Reagents supplied: 10x NotI buffer

Unit definition: One unit is the amount of enzyme required to digest 1 μg of Adenovirus-2 DNA in 60 min at 37oC in a total reaction volume of 50 μl.

(SKUs: RE025S, RE025L, REc025S, REc025L)

60.00144.00
  • 10u/μl
  • 40-60u/μl
  • 500 U
  • 3×500 U

Product Details

NotI is a restriction enzyme purified from Nocardia otitidis-caviarum.

Reagents supplied: 10x NotI buffer

Unit definition: One unit is the amount of enzyme required to digest 1 μg of Adenovirus-2 DNA in 60 min at 37oC 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 Not I1μl
10x NotI buffer5μl
DNA substrate1μg
Sterile ultrapure waterUp to 50 μl
  • Speed Digest reaction: Incubate for 15 minutes at 37oC as in standard reaction in 1x NotI buffer (100 mM NaCl, 50 mM Tris-HCl (pH 7.9 @ 25°C), 5 mM MgCl2, 100 μg/ml BSA).
  • Activity (%) in EnzyQuest buffers -Double digest tips
R1R2R3R4EQ
<1025-5075-1007550

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

Methylation Sensitivity:

  • dam methylation: Not sensitive
  • dcm methylation: Not sensitive
  • CpG methylation: Blocked

Unit calculation assay conditions:

  • 1x NotI buffer
  • Adenovirus-2 DNA
  • Incubation at 37oC

Storage conditions:

  • 500 mM KCl,
  • 10 mM Tris-HCl (pH 7.4)
  • 0.1 mM EDTA
  • 1 mM dithiothreitol
  • 0.1% Triton X-100
  • 500 μg/ml BSA
  • 50% glycerol
  • Store at -20oC

Non-specific nuclease activity: 80 units of Not I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Adeno-2 DNA at 37°C.

Re-ligation and recut: After 30-fold overdigestion with Not I, greater than 98% of the DNA fragments can be ligated and recut with this enzyme.

Note: Supercoiled plasmids may require up to 5-fold more Not I for complete digestion than linear DNAs.

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