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Not I

from Nocardia otitidis-caviarum

Quality Level




pkg of 1,000 U (11014714001 [10 U/μl])
pkg of 1,000 U (11037668001 [40 U/μl])
pkg of 200 U (11014706001 [10 U/μl])




37 °C optimum reaction temp.


PCR: suitable

shipped in

dry ice

storage temp.


General description

Not I recognizes the sequence GC?GG*C*CG°C and generates fragments with 5′-cohesive termini.
Not I belongs to the class of "rare-cutter" enzymes. It is one of the two known enzymes that recognize an octameric sequence comprised solely of G and C residues.

  • Not I
  • SuRE/Cut Buffer H (10x)


Recognition sites: GCGG*C*CG °C
Restriction site: GC↓GG*C*CG °C
Heat inactivation: Not I can be heat inactivated by incubation at 65 °C for 15 minutes (up to 100 U/μg DNA).


Absence of nonspecific endonuclease activities
1 μg Ad2 DNA is incubated for 16 hours in 50 μl SuRE/Cut Buffer H with an excess of Not I. The number of enzyme units which do not change the enzyme-specific pattern is stated in the certificate of analysis.

Absence of exonuclease activity

Approximately 5 μg [3H] labeled calf thymus DNA are incubated with 3 μl Not I for 4 hours at +37°C in a total volume of 100 μl 50 mM Tris-HCl, 10 mM MgCl2, 1 mM Dithioerythritol, pH approximately 7.5. Under these conditions, no release of radioactivity is detectable, as stated in the certificate of analysis.


Average size of fragment generated
Prokaryotic genomic DNA: Not I fragments are between 20 and 1,000 kb, depending on the GC content.
Yeast genomic DNA: Not I fragments are, on average, 200 kb.
Mammalian genomic DNA: Not I fragments are approximately 1,000 kb.

Compatible ends
Not I ends are compatible with ends generated by Eae I and EclX I (Xma III).

The enzyme has no known isoschizomers.

Methylation sensitivity
Not I is inhibited by the presence of 5-methylcytosine at the sites indicated (*) on the recognition sequence. However, the presence of 5-methylcytosine in the 5′-C position (°) is not inhibiting.

Relative activity in complete PCR mix
Relative activity in PCR mix (Taq DNA Polymerase buffer) is 0%. The PCR mix contained λDNA, primers, 10 mM Tris-HCl (pH 8.3, +20°C), 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTPs, 2.5 U Taq DNA polymerase. The mix was subjected to 25 amplification cycles. After addition of 100 mM NaCl to the RE digest in the PCR mix, the activity of Not I still remains very low with below 5%.

Incubation temperature

PFGE tested
Not I has been tested in Pulsed-Field Gel Electrophoresis (on bacterial chromosomes). For cleavage of genomic DNA (E. coli C 600) embedded in agarose for PFGE analysis, we recommend using 10 U of enzyme/μg DNA and 4 hour incubation time.

Ligation and recutting assay
Not I fragments obtained by complete digestion of 1 μg Ad2 DNA are ligated with 1 U T4 DNA Ligase in a volume of 10 μl by incubation for 16 hours at +4°C in 66 mM Tris-HCl, 5 mM MgCl2, 5 mM Dithiothreitol, 1 mM ATP, pH 7.5 (at +20°C) resulting in >80% recovery of Ad2 DNA.
Subsequent re-cutting with Not I yields >90% of the typical pattern of Ad2 × Not I fragments.

DNA Profile

Number of cleavage sites on different DNAs
  • λ: 0
  • φX174: 0
  • Ad2: 7
  • M13mp7: 0
  • pBR322: 0
  • pBR328: 0
  • pUC18: 0
  • SV40: 0

Unit Definition

One unit is the enzyme activity that completely cleaves 1 μg Ad2DNA in one hour at +37 °C in a total volume of 25 μl (1x) SuRE/Cut Buffer H. The 8 fragments obtained are 18629, 6493, 5001, 2594, 1931, 954, 326 and 9 bp in length. Ad2 DNA has one Not I cleavage site that is cleaved much more slowly than the other 6 cleavage sites.

Analysis Note

SuRE/Cut Buffer System
The buffer in bold is recommended for optimal activity
  • A: 10-25%
  • B: 50-75%
  • H: 100%
  • L: 0-10%
  • M: 25-50%

Activity in PCR buffer: 0%

Other Notes

For life science research only. Not for use in diagnostic procedures.

Kit Components Only

Product No.

  • Enzyme Solution

  • SuRE/Cut Buffer H 10x concentrated

Storage Class Code

12 - Non Combustible Liquids



Flash Point(F)

does not flash

Flash Point(C)

does not flash

Certificate of Analysis

Certificate of Origin


Restriction Endonucleases - The Molecular Scissors

The term “Restriction enzyme” originated from the studies of Enterobacteria phage λ (lambda phage) in the laboratories of Werner Arber and Matthew Meselson.

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Restriction Enzymes

Restriction endonucleases popularly referred to as restriction enzymes, are ubiquitously present in prokaryotes. The function of restriction endonucleases is mainly protection against foreign genetic material especially against bacteriophage DNA.

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