Dpn I
ROCHE/DPNI-RO - from Diplococcus pneumoniae
Product Type: Chemical
| biological source | bacterial (Diplococcus pneumoniae) |
| form | solution |
| manufacturer/tradename | Roche |
| packaging | pkg of 1,000 U (10742988001 [10 U/μl]) |
| pkg of 200 U (10742970001 [10 U/μl]) | |
| parameter | 37 °C optimum reaction temp. |
| Quality Level | 100  |
| storage temp. | −20°C |
| Analysis Note: | SuRE/Cut Buffer System The buffer in bold is recommended for optimal activity • A: 100% • B: 75-100% • H: 75-100% • L: 50-75% • M: 75-100% |
| Analysis Note: | Activity in PCR buffer: 100% Relative activity in PCR mix (Taq DNA Polymerase buffer) is 100%. 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.Activity in reaction buffer of Pwo SuperYield DNA Polymerase PCR Mix is 100%; Pwo SuperYield DNA Polymerase PCR Mix is not available in U.S. |
| Application: | In PCR-mediated mutagenesis, DpnI nuclease has been used for the digestion of wild type DNA after PCR. |
| Compatibility: | Dpn I generates fragments with blunt ends that are compatible with any blunt end. |
| DNA Profile: | Number of cleavage sites on different DNAs • λ: 116 • φX174: 0 • Ad2: 87 • M13mp7: 8 • pBR322: 22 • pBR328: 27 • pUC18: 15 • SV40: 8 |
| General description: | Isoschizomers The enzyme is an isoschizomer to Bsp143 I, Dpn II, Mbo I, Nde II and Sau3A I. Methylation sensitivity The enzyme is only inhibited by the occurrence of 5-methylcytosine at the indicated site (*) if no 6-methyladenine is present. Typical ligation and recutting assay Dpn I fragments obtained by complete digestion of 1μg pBR322 DNA are ligated for 16 hours at +4°C with 1U T4 DNA Ligase in 10μl buffer that contains 66mM Tris-HCl, 5mM MgCl2, 5mM Dithiothreitol, 1mM ATP, pH 7.5 (at +20°C). The percentages of product that can be ligated and subsequently recut with Dpn I (yielding the typical pattern of pBR322 × Dpn I fragments) are stated under "Lig" and "Rec" in the certificate of analysis. |
| General description: | Dpn I requires methylation of adenine residues for activity and thus digests only GmATC sequences containing N6-methyladenine. Such methylated sequences are produced by dam methylase. This requirement for methylation distinguishes Dpn I from Mbo I, which is inhibited by dam methylation, and Sau3A I, which is not influenced by dam methylation. Dpn I also cleaves at a different position on the recognition sequence than Mbo I and Sau3A I. |
| Other Notes: | For life science research only. Not for use in diagnostic procedures. |
| Quality: | Absence of nonspecific endonuclease activities 1μg pBR322 DNA is incubated for 16 hours in 50μl SuRE/Cut Buffer A with an excess of Dpn 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 Dpn I for 4 hours 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: | Recognition sites: GmAT*C GmAT*C Restriction site: GmA↓T*C GmA↓T*C Heat inactivation: No inactivation of Dpn I after incubation at 65 °C for 15 minutes. |
| Unit Definition: | One unit is the enzyme activity that cleaves 1 μg pBR322 DNA in one hour at +37 °C in a total volume of 25 μl (1x) SuRE/Cut Buffer A. Since complete Dpn I digestion of pBR322 DNA needs fully methylated GATC sequences < 5% partial digested bands may be observed during activity determination. |
| RIDADR | NONH for all modes of transport |
| WGK Germany | WGK 1 |
| Flash Point(F) | does not flash |
| Flash Point(C) | does not flash |
| Storage Temp. | −20°C |
| UNSPSC | 12352204 |
| Components | Enzyme Solution; SuRE/Cut Buffer A 10x concentrated |