New England Biolabs, M0646T, EnGen® Spy Cas9 NLS

Now available: EnGen Spy Cas9 HF1 Related Categories CRISPR/Cas Nucleases Specification 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: Where is the nuclear localization signal on EnGen® Spy Cas9 NLS located? A: The EnGen Spy Cas9 NLS contains two nuclear localization signals located on the N- and C- termini of the protein. Q: Which nuclear localization signal is fused to EnGen® Spy Cas9 NLS? A: EnGen Spy Cas9 NLS contains Simian virus 40 (SV40) T antigen nuclear localization signal (NLS) on the N- and C- termini of the protein. Q: What is the difference between EnGen® Spy Cas9 NLS (NEB #M0646) and Cas9 NLS, S.pyogenes (NEB #M0641)? A: EnGen Spy Cas9 NLS (NEB #M0646) contains two nuclear localization signals located on the N- and C- termini of the protein. In contrast Cas9 NLS, S. pyogenes (NEB #M0641), contains one NLS signal located on the C- terminus of the protein. 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: 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: 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.