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Analysis Note

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.

Compatible endsPvu I generates ends that are compatible with fragments generated by Pac I.IsoschizomersPvu I is an isoschizomer to BspC I and Xor II.Methylation sensitivityPvu 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 SystemThe 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°CUnit definitionOne 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 inactivationPvu I cannot be heat inactivated by incubating it for 15 minutes at +65°C.PFGE testedPvu 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 assayPvu 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.

DNA Profile

Number of cleavage sites on different DNAs λ: 3 φX174: 0 Ad2: 7 M13mp7: 1 pBR322: 1 pBR328: 1 pUC18: 2 SV40: 0

Other Notes

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

Quality

Absence of nonspecific endonuclease activities1µg 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 activityApproximately 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.

Specificity

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

Unit Definition

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.