Not I

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.

Contents:

• Not I
• SuRE/Cut Buffer H (10x)

Recognition sites: GCGG*C*CG °C
GCGG*C*CG °C
Restriction site: GC↓GG*C*CG °C
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).

Isoschizomers
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
+37°C

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.

Number of cleavage sites on different DNAs

• λ: 0
• φX174: 0
• Ad2: 7
• M13mp7: 0
• pBR322: 0
• pBR328: 0
• pUC18: 0
• SV40: 0

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.

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%

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