New England Biolabs, R0176S, DpnI

DpnI has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10196884. Related Categories Methylation Sensitive and Methylation Dependent Restriction Enzymes for Epigenetics,, Restriction Enzymes for Epigenetic Analysis,, Restriction Endonucleases C G, Applications Site Directed Mutagenesis,, Site Directed Mutagenesis,, Fast Cloning: Accelerate your cloning workflows with reagents from NEB, Specification Unit Definition One unit is defined as the amount of enzyme required to digest 1 µg of pBR322 DNA ( dam methylated) in 1 hour at 37°C in a total reaction volume of 50 μl. Reaction Conditions 1X rCutSmart™ Buffer Incubate at 37°C 1X rCutSmart™ Buffer 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Activity in NEBuffers NEBuffer™ r1.1: 100% NEBuffer™ r2.1: 100% NEBuffer™ r3.1: 75% rCutSmart™ Buffer: 100% Diluent Compatibility Diluent B Storage Buffer 10 mM Tris-HCl 300 mM NaCl 1 mM DTT 0.1 mM EDTA 500 µg/ml Recombinant Albumin 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 80°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Not Sensitive CpG Methylation: Blocked by Overlapping FAQ Q: Will DpnI cleave hemimethylated DNA? A: DpnI cleaves hemi-methylated dam sites 60X more slowly than fully methylated. Q: What's the difference between DpnI, DpnII, MboI, and Sau3AI? A: They all recognize the same sequence but have different methylation sensitivities. DpnI will only cleave fully-adenomethylated dam sites and hemi-adenomethylated dam sites 60X more slowly. DpnII and MboI share methylation sensitivity and cleave dam sites which lack adenomethylation; each is blocked by complete dam methylation and probably by hemi-methylation as well. Sau3AI will cleave all dam sites regardless of adenomethylation but is completely blocked by methylated cytosines within the dam sequence. Q: Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer? A: NEB's Time-Saver™ enzymes have the benefit of working fast (5-15 minutes), but are also designed and qualified to withstand overnight digestions without degradation of DNA. Q: Which NEB restriction enzymes are supplied with Gel Loading Dye, Purple (6X)? A: All HF-restriction enzymes and most NEB non-HF restriction enzymes are supplied with Gel Loading Dye, Purple (6X). The non-HF restriction enzymes that come supplied with purple dye are: AatII AsiSI BsmBI-v2 EagI MboI NlaIII PvuI SmaI Acil AvrII BspHI EcoRI MboII NotI RsaI SpeI AfeI BamHI BsrGI EcoRV MluI NspI SacI StuI AflII BbsI ClaI Esp3I FseI MseI PacI SacII AgeI BglII DdeI HaeIII MspI PciI SalI XbaI AluI BsaI DpnI HindIII NcoI PmeI SapI XhoI ApaI BseYI DpnII HpaI NdeI PsiI-v2 SfaNI XmaI ApeKI BsiWI DraI KpnI NheI PstI SfiI XmnI AscI * All HF restriction enzymes are also supplied with Gel Loading Dye, Purple (6X) Q: Is this enzyme sensitive to dam, dcm or mammalian CpG methylation? A: Yes. This enzyme cannot cut recognition sites in mammalian gDNA that are blocked by overlapping CpG methylation. For up-to-date information about methylation sensitivities, please visit Dam-Dcm and CpG Methylation or REBASE. Q: Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers? A: NEB is excited to announce that we have switched BSA-containing reaction buffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We are also in the process of moving all restriction enzyme formulations to contain rAlbumin. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price.