New England Biolabs, M0652T, EnGen® Spy dCas9 SNAP tag

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 FAQ Q: Where is the nuclear localization signal on SNAP dCas9 NLS, S. pyogenes located? A: SNAP dCas9 NLS, S. pyogenes contains one nuclear localization signal located on the C- terminus of the protein. Q: Which nuclear localization signal is fused to SNAP dCas9 NLS, S. pyogenes? A: SNAP dCas9 NLS, S. pyogenes contains a Simian virus 40 (SV40) T antigen nuclear localization signal (NLS). Q: What inactivating mutations were made in SNAP dCas9 NLS, S. pyogenes? A: SNAP dCas9 NLS, S. pyogenes contains two point mutations, D10A and H840A, that inactivate the nuclease domains of SpCas9. Q: Why do I observe incomplete binding with SNAP dCas9 NLS, S. pyogenes? A: Incomplete binding may be due to the following factors: Incorrect ratio of dCas9 to guide RNA, and target site-For complete binding we recommend a 10:10:1 or higher molar ratio (SNAP dCas9 NLS : guide RNA : target site). Suboptimal guide RNA sequence-Verify the sequence and design of the guide RNA. Poor quality guide RNA-Verify the integrity of the guide RNA by gel electrophoresis. Buffer incompatibility-Please use the 10X NEBuffer 3.1 included with the enzyme. Q: Why does binding efficiency differ between two different guide RNAs? A: Binding efficiency may be influenced by guide RNA design. Verify the sequence and design of the guide RNA transcription template. It may also be influenced by guide RNA quality. Verify the integrity of the guide RNA by gel electrophoresis. 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: 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.