{"product_id":"neb-m0668t","title":"New England Biolabs, M0668T, EnGen® Seq1 Cas9","description":"\u003cb\u003eRelated Categories\u003c\/b\u003e CRISPR\/Cas Nucleases  \u003cb\u003eSpecification\u003c\/b\u003e \u003cb\u003eReaction Conditions\u003c\/b\u003e 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)  \u003cb\u003eStorage Buffer\u003c\/b\u003e 300 mM NaCl 10 mM Tris-HCl 0.1 mM EDTA 1 mM DTT 50% Glycerol pH 7.4 @ 25°C  \u003cb\u003eHeat Inactivation\u003c\/b\u003e 65°C for 5 minutes  \u003cb\u003eFAQ\u003c\/b\u003e Q: Where is the nuclear localization signal on EnGen® Seq1 Cas9 located? A: EnGen Seq1 Cas9 contains two nuclear localization signals located on the N- and C-termini of the protein. Q: Which nuclear localization signal is fused to EnGen® Seq1 Cas9? A: EnGen Seq1 Cas9 contains Simian virus 40 (SV40) T antigen nuclear localization signal on the N- and C-termini of the protein. Q: What is the difference between EnGen® Seq1 Cas9, EnGen Sau Cas9 (NEB #M0654), and EnGen Spy Cas9 NLS (NEB #M0646)? A: EnGen Seq1 Cas9, EnGen Sau Cas9, and EnGen Spy Cas9 NLS are Cas9 orthologs from Streptococcus equinus, Staphylococcus aureus, and Streptococcus pyogenes, respectively. They recognize the protospacer adjacent motifs (PAMs) of NAGA, NNGRRT, and NGG, respectively. Q: Why do I observe incomplete digestion\/editing with  EnGen® Seq1 Cas9? A: Incomplete digestion may be due to the following factors: Suboptimal ratio of Cas9 nuclease to single guide RNA and DNA target site – For complete digestion we recommend a 10:10:1 or higher molar ratio of Cas9 nuclease: single guide RNA: DNA target. Suboptimal sequence of the single guide RNA – Verify the sequence and design of the single guide RNA. Poor quality single guide RNA – Verify the integrity of the single guide RNA by gel electrophoresis. Suboptimal reaction buffer – Please use the NEBuffer™ r3.1 included with the enzyme. Q: Why does digestion\/editing efficiency differ between two different single guide RNAs? A: Digestion efficiency may be influenced by single guide RNA design. Verify the sequence and design of the single guide RNA transcription template. It may also be influenced by single guide RNA quality. Verify the integrity of the single guide RNA by gel electrophoresis. Additionally, some DNA sites are more amenable to editing than others. Please test multiple target sites\/single guide RNA sequences appropriate for the desired mutation. Q: Does NEB provide plasmids for sgRNA cloning? A: We do not distribute plasmids for sgRNA cloning, but we recommend that you visit Addgene if you wish to obtain sgRNA plasmids. DNA templates containing a T7 promoter and encoding a sgRNA can be used with the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB #E2050). Additionally, there are many available commercial sources of synthetic sgRNA. 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. Q: Can mutations generated with EnGen Seq1 Cas9 be detected using T7 Endonuclease I (NEB #M0302) or the EnGen Mutation Detection Kit (NEB #E3321S)? A: Yes, both of these products can be used to detect genome editing events generated with EnGen Seq1 Cas9. Q: Can sgRNA for use with EnGen Seq1 Cas9 be generated using the EnGen sgRNA Synthesis Kit, S. pyogenes (NEB #E3322)? A: No, the EnGen sgRNA Synthesis Kit, S. pyogenes cannot be used to make sgRNAs for EnGen Seq1 Cas9. The scaffold oligo in the 2X sgRNA Reaction Mix scaffold DNA, which contains the tracrRNA sequence, is only compatible with S. pyogenes Cas9. Q: How do I design a single guide RNA for use with EnGen Seq1 Cas9? A: The single guide RNA for use with EnGen Seq1 Cas9 has the following sequence: 5’- NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUGUGUUGGAAACAACACAGCGAGUUAAAAUAAGGCUUUGUCCGUACACAACUUGUAAAAGUGGCACCCGAUUCGGGUGCAUUUUUU -3’ The underlined 20-nucleotide spacer sequence is specific to the desired DNA target region immediately upstream of the 5’-NAGA-3’ protospacer adjacent motif. The spacer sequence does not contain the PAM sequence itself. The portion of the sgRNA not underlined is the scaffold specific for ribonucleoprotein complex formation with EnGen Seq1 Cas9. 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: Why do I observe incomplete digestion\/editing with EnGen® Spy Cas9 HF1? 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™ r3.1 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: What is the difference between EnGen® Spy Cas9 HF1 and EnGen Spy Cas9 NLS (NEB #M0646)? A: EnGen Spy Cas9 HF1 is a high-fidelity, quadruple substitution (N497A\/R661A\/Q695A\/Q926A) variant of EnGen Spy Cas9 NLS from Streptococcus pyogenes with reduced non-specific DNA cleavage. Q: Where is the nuclear localization signal on EnGen® Spy Cas9 HF1 located? A: EnGen Spy Cas9 HF1 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 HF1? A: EnGen Spy Cas9 HF1 contains Simian virus 40 (SV40) T antigen nuclear localization signal (NLS) on the N- and C- termini of the protein. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835528204457,"sku":"M0668T","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-m0668t","provider":"Iright","version":"1.0","type":"link"}