Nde I

Recognition sites: °CAT*ATG
°CAT*ATG
Restriction site: °CA↓T*ATG
°CA↓T*ATG
Heat inactivation: Nde I can be heat inactivated by incubation at 65 °C for 15 minutes.

Methylation sensitivity: Nde I is inhibited by internal 6-methyladenine at the site indicated (*) on the recognition sequence. The enzyme is not inhibited by 5-methylcytosine (°).

Absence of nonspecific endonuclease activities: 1 μg λDNA is incubated for 16 hours in 50 μl SuRE/Cut Buffer H with an excess of Nde 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 Nde 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.

Compatible ends: Nde I ends are compatible with ends generated by Asn I, Mae I, and Tru9 I.

Isoschizomers: The enzyme has no known isoschizomers.

Number of cleavage sites on different DNAs

• λ: 7
• φX174: 0
• Ad2: 2
• M13mp7: 3
• pBR322: 1
• pBR328: 0
• pUC18: 1
• SV40: 2

One unit is the enzyme activity that completely cleaves 1 μg λDNA in one hour at +37 °C in a total volume of 25 μl (1x) SuRE/Cut Buffer H.

Ligation and recutting assay:
Nde I fragments obtained by complete digestion of 1 μg λDNA 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 >95% recovery of 1 μg λDNA fragments.
Subsequent re-cutting with Nde I yields >95% of the typical pattern of λDNA × Nde I fragments.
Incubation temperature:+37°C

SuRE/Cut Buffer System
The buffer in bold is recommended for optimal activity

• A: 25-50%
• B: 75-100%
• H: 100%
• L: 10-25%
• M: 50-75%

Activity in PCR buffer: 0%

Relative activity in PCR mix (Taq DNA Polymerase buffer) is 0%. The PCR mix contained target DNA, 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.Activity in reaction buffer of Pwo SuperYield DNA Polymerase PCR Mix is 40%. When supplemented with GC-RICH Solution activity is increased up to 100%.

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