Kpn I

Compatible ends
Kpn I ends are not compatible with those generated by any other known restriction enzymes.

Isoschizomers
Kpn I is an isoschizomer of Asp 718 I (which generates fragments with 5′-cohesive termini).

Methylation sensitivity
Kpn I is not inhibited by 5-methylcytosine at either or both C-residues in the recognition sequence, nor is it inhibited by 6-methyladenine.

Ligation and recutting assay
Kpn I fragments obtained by complete digestion of 1 μg λ × EcoR I fragments 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 × EcoR I fragments.
Subsequent re-cutting with Kpn I yields >95% of the typical pattern of λDNA × EcoR I × Kpn I fragments.

The recognition specifiity of Kpn I is altered by addition of increasing amounts of hydrophobic reagents and glycerol to the incubation mixture.
Recognition sites: GGTACC
GGTACC
Restriction site: GGTAC↓C
GGTAC↓C
Heat inactivation: Kpn I can be heat inactivated by incubation at 65 °C for 15 minutes (up to 3 U/μg DNA).
Star activity: Kpn I may exhibit star activity under non-optimal conditions.

SuRE/Cut buffer L requires addition of BSA (100 μg/ml final concentration).

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

Number of cleavage sites on different DNAs

• λ: 2
• φX174: 0
• Ad2: 8
• M13mp7: 0
• M13mp18: 1
• pBR322: 0
• pBR328: 0
• pUC18: 1
• SV40: 1

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 L, including 100 μg/ml BSA.

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

• A: 75-100%
• B: 10-25%
• H: 0-10%
• L: 100%*
• M: 25-50%

* Supplied SuRE/Cut Buffer L requires the addition of BSA (100 μg/ml final concentration).

Activity in PCR buffer: 50%

Relative activity in PCR mix (Taq DNA Polymerase buffer) is 50%. 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 100%. When supplemented with GC-RICH Solution: activity remains at 100%. Pwo SuperYield DNA Polymerase PCR Mix is not available in U.S.

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