New England Biolabs, R0140T, PstI

Related Categories Restriction Endonucleases P R,, Time-Saver Qualified Restriction Enzymes Applications BioBrick, ®, Assembly,, Fast Cloning: Accelerate your cloning workflows with reagents from NEB Specification Unit Definition One unit is defined as the amount of PstI required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 μl. Reaction Conditions 1X NEBuffer™ r3.1 Incubate at 37°C 1X NEBuffer™ r3.1 100 mM NaCl 50 mM Tris-HCl 10 mM MgCl2 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Activity in NEBuffers NEBuffer™ r1.1: 75% NEBuffer™ r2.1: 75% NEBuffer™ r3.1: 100% rCutSmart™ Buffer: 50% Diluent Compatibility Diluent C Storage Buffer 250 mM NaCl 10 mM Tris-HCl 1 mM DTT 0.1 mM EDTA 200 µg/ml Recombinant Albumin 50% Glycerol 0.15% Triton® X-100 pH 7.4 @ 25°C Heat Inactivation 80°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Not Sensitive CpG Methylation: Not Sensitive FAQ Q: What is the activity of PstI at 25°C? A: PstI is 50-75% active at 25°C. Q: Is PstI used in special techniques? A: Yes, PstI is used for DNA typing. Q: When is star activity a problem for PstI? A: Conditions of high pH, low salt, high glycerol; 8% DMSO can cause star activity. (Malyguine et al. (1980). Gene 8: 163-177). Q: What is the molecular weight of PstI? A: The molecular weight of PstI is 37.37 kDa based on sequence data. Q: Is PstI blocked by methylation? A: PstI is not blocked by Dam, Dcm or CpG methylation. However PstI is blocked by certain types of methylation sometimes found in plant DNA. Plants often have CAG and/or CTG methylation leading to MTG and/or MAG sites. Where M is 5mC. Pst I is blocked by this type of methylation. This is not the same as CpG methylation (MG as opposed to MTG and MAG). For up-to-date information about methylation sensitivities, please visit Dam-Dcm and CpG Methylation or REBASE. Pst I will also not cut AGCTGCAG when methylated by AluI methylase. Q: Is PstI inhibited by dUTP incorporated at the site? A: Yes. 100% dUTP incoporation reduces PstI cleavage to 25%. Glenn et al. (1994). Biotechniques 17: 1086-1090. Q: Does spermidine increase activity? A: No, 0.5-2mM spermidine inhibits digestion by PstI (Kuosmanen and Poso (1985). FEBS Lett. 179: 17-20. Spermidine suppresses star activity of PstI (Pingoud (1985). Eur. J. Biochem. 147:150-109. Q: Are there specific surrounding sequences that produce slow or resistant sites? A: The presence of adjacent runs of G-C base pairs confers significant resistance to cleavage. Armstrong and Bauer (1982). NAR 10: 993-1007. 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: 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.