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Analysis Note

Activity in PCR buffer: 0%

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

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 activities1 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 activityApproximately 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 generatedProkaryotic 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 endsNot I ends are compatible with ends generated by Eae I and EclX I (Xma III).IsoschizomersThe enzyme has no known isoschizomers.Methylation sensitivityNot 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 mixRelative 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°CPFGE testedNot 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 assayNot 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 °CGCGG*C*CG °CRestriction site: GC↓GG*C*CG °CGC↓GG*C*CG °CHeat 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.