New England Biolabs, M0386M, Cas9 Nuclease, S. pyogenes

Now available: EnGen Spy Cas9 HF1 Related Categories Validation of CRISPR-based Gene Editing,, CRISPR/Cas Nucleases Specification Materials Required but not Supplied User-supplied sgRNA EnGen® sgRNA Synthesis Kit, S. Pyogenes (NEB #E3322) HiScribe® Quick T7 High Yield RNA Synthesis Kit (NEB #E2050) Outside vendor Nuclease-free Water (NEB #B1500) Proteinase K, Molecular Biology Grade (NEB #P8107). 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) Storage Buffer 300 mM NaCl 10 mM Tris-HCl 0.1 mM EDTA 1 mM DTT 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 5 minutes FAQ Q: Why do I observe incomplete digestion? A: Incomplete digestion may be due to the following factors: Incorrect ratio of Cas9 Nuclease to guide RNA, and target site- For complete digestion we recommend a 10:10:1 or higher molar ratio of Cas9 Nuclease: guide RNA : target site. Suboptimal sequence of the guide RNA- Verify the sequence and design of the guide RNA. Poor quality guide RNA- Verify the integrity of the guide RNA by gel electrophoresis. Suboptimal buffer- Please use the NEBuffer 3.1 included with the enzyme. Q: Why does digestion efficiency differ between two SgRNAs? A: Digestion efficiency may be influenced by sgRNA design. Verify the sequence and design of the sgRNA transcription template. Digestion efficiency may be influenced by sgRNA quality. Verify the integrity of the sgRNA by gel electrophoresis. Q: Does NEB provide plasmids for gRNA cloning? A: We do not distribute plasmids for sgRNA cloning, but we recommend that you visit Addgene if you wish to obtain sgRNA plasmids. Oligonucleotide DNA templates containing a T7 promoter and encoding sgRNA can be used with the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB #E2050). We recommend using the EnGen® sgRNA Synthesis Kit, S. pyogenes (NEB #E3322S), which utilizes target-specific DNA oligos designed by the user and provides a quick method for transcribing high yields of sgRNA in a single 30 minute reaction. Q: Does Cas9 Nuclease contain a nuclear localization signal (NLS)? A: No, Cas 9 Nuclease (NEB #M0386) does not have a nuclear localization signal. We do offer an alternative product, EnGen® Cas9 NLS, S. pyogenes (NEB# M0646) with nuclear localization signal. Q: Is Cas9 Nuclease fused with a N-terminal 6XHis tag? A: Yes Q: How do I dilute the enzyme to 1 μM for in vitro reactions? A: If planning to use the higher concentration enzyme for in vitro digestion of DNA, the enzyme can be diluted to 1 μM in 1X NEBuffer™ r3.1 and used immediately. The 1 μM dilution in 1X NEBuffer r3.1 should not be frozen. If the 1 μM dilution will be stored at -20°C, it should be diluted using Diluent B (with rAlbumin) (NEB #B8533S): 300 μM NaCl, 10 μM Tris-HCl, 0.1 μM EDTA, 1 μM DTT, 500 μg/ml Recombinant Albumin and 50% glycerol (pH 7.4 @ 25°C) prior to the reaction assembly. 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.