{"product_id":"neb-m0667t","title":"New England Biolabs, M0667T, EnGen® Spy Cas9 HF1","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® 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. 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: 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: 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. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835494322345,"sku":"M0667T","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-m0667t","provider":"Iright","version":"1.0","type":"link"}