Not I
ROCHE/NOTI-RO - from Nocardia otitidis-caviarum
Synonym: restriction enzyme
Product Type: Chemical
| biological source | bacterial (Nocardia otitidis-caviarum) |
| form | solution |
| manufacturer/tradename | Roche |
| packaging | 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]) | |
| parameter | 37 °C optimum reaction temp. |
| Quality Level | 100  |
| storage temp. | −20°C |
| technique(s) | PCR: suitable |
| 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% |
| Analysis Note: | Activity in PCR buffer: 0% |
| 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 |
| 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. Contents: • Not I • SuRE/Cut Buffer H (10x) |
| Other Notes: | For life science research only. Not for use in diagnostic procedures. |
| Quality: | 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. |
| Specifications: | 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. |
| Specificity: | 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). |
| 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. |
| RIDADR | NONH for all modes of transport |
| WGK Germany | WGK 1 |
| Flash Point(F) | does not flash |
| Flash Point(C) | does not flash |
| Storage Temp. | −20°C |
| UNSPSC | 12352204 |
| Components | Enzyme Solution; SuRE/Cut Buffer H 10x concentrated |