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Analysis Note

SuRE/Cut Buffer SystemThe buffer in bold is recommended for optimal activity A: 100% B: 75-100% H: 75-100% L: 50-75% M: 75-100%

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

IsoschizomersThe enzyme is an isoschizomer to Bsp143 I, Dpn II, Mbo I, Nde II and Sau3A I.Methylation sensitivityThe enzyme is only inhibited by the occurrence of 5-methylcytosine at the indicated site (*) if no 6-methyladenine is present.Typical ligation and recutting assayDpn 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.

Dpn I requires methylation of adenine residues for activity and thus digests only G^mATC 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 activities1µg 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 activityApproximately 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*CG^mAT*CRestriction site: GmA↓T*CG^mA↓T*CHeat 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.