Pvu I

Number of cleavage sites on different DNAs

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

Pvu I recognizes the sequence CG°AT↓ ^*CG and generates fragments with 3′-cohesive termini.
Recognition sites: CG°AT*CG
CG°AT*CG
Restriction site: CG°AT↓*CG
CG°AT↓*CG
Heat inactivation: No inactivation of Pvu I after incubation at 65 °C for 15 minutes.

Do not store below −25°C.

Absence of nonspecific endonuclease activities
1μg λDNA is incubated for 16hours in 50μl SuRE/Cut Buffer H with an excess of Pvu 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 5μg [3H] labeled calf thymus DNA are incubated with 3μl Pvu I for 4hours at +37°C in a total volume of 100μl 50mM Tris-HCl, 10mM MgCl₂, 1mM Dithioerythritol, pH approximately 7.5. Under these conditions, no release of radioactivity is detectable, as stated in the certificate of analysis.

Compatible ends
Pvu I generates ends that are compatible with fragments generated by Pac I.

Isoschizomers
Pvu I is an isoschizomer to BspC I and Xor II.

Methylation sensitivity
Pvu I cleavage is not inhibited by overlapping dam-methylation at the site indicated (°) on the recognition sequence, but Pvu I fragments of DNA isolated from dam+ strains are not as readily religated as those isolated from dam- strains. Pvu I is inhibited by 5-methylcytosine at the indicated site (°) and by 4-methylcytosine.

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

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

Incubation temperature
+37°C

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

Heat inactivation
Pvu I cannot be heat inactivated by incubating it for 15 minutes at +65°C.

PFGE tested
Pvu 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 10U of enzyme/μg DNA and 4 hour incubation time.

Ligation and recutting assay
Pvu I fragments obtained by complete digestion of 1 μg pBR322 DNA are ligated with 1U T4 DNA Ligase in a volume of 10μl by incubation for 16hours at +4°C in 66mM Tris-HCl, 5mM MgCl₂, 5mM Dithiothreitol, 1mM ATP, pH 7.5 (at +20°C) resulting in >80% recovery of 1μg pBR322 DNA fragments.
Subsequent re-cutting with Pvu I yields >95% of the typical pattern of pBR322 DNA × Pvu I fragments.

Activity in PCR buffer: <5%

Relative activity in PCR mix (Taq DNA Polymerase buffer) is less than 5%. 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.

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

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