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.
Standard reaction conditions: In order to overcome DNA source variability, a ~10 fold overdigest is suggested:
10U Not I
10x NotI buffer
Sterile ultrapure water
Up 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
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
Unit calculation assay conditions:
1x NotI buffer
Incubation at 37oC
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
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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