New England Biolabs, R0725S, WarmStart® Nt.BstNBI

Related Categories Nicking Endonucleases,, Restriction Endonucleases: N-O,, Restriction Endonucleases Applications Strand Displacement Amplification & Nicking Enzyme Specification Unit Definition One unit is defined as the amount of enzyme required to digest 1 µg T7 DNA in NEBuffer r3.1 in 1 hour at 55°C in a total reaction volume of 50 μl. Reaction Conditions 1X NEBuffer™ r3.1 Incubate at 55°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) Usage Concentration 10X Activity in NEBuffers NEBuffer™ r1.1: 0% NEBuffer™ r2.1: 10% NEBuffer™ r3.1: 100% rCutSmart™ Buffer: 25% Diluent Compatibility Diluent A Storage Buffer 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 200 µg/ml Recombinant Albumin 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 80°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Not Sensitive CpG Methylation: Not Sensitive Activity at Temperature @37°C: 0% FAQ Q: What advantages does WarmStart® Nt.BstNBI provide? A: WarmStart® Nt.BstNBI can be used in applications that Nt.BstNBI is used. The aptamer in WarmStart Nt.BstNBI inhibits nicking activity below 40°C, therefore the enzyme only becomes fully activated when reaction temperatures of 50-60°C are reached. This feature allows high-throughput assay assembly and room temperature setup (such as SDA). In addition, since the enzyme activity is precisely controlled, the results are consistent and variations between replicates are low. Q: What is Strand Displacement Amplification (SDA)? A: Strand displacement amplification is an isothermal in vitro DNA amplification method that relies on a nicking enzyme to nick one strand of double-stranded DNA and a DNA polymerase with exonuclease deficiency to extend the 3´-end at the nick and displace the downstream DNA strand (GT Walker, 1992). This method can be applied to rapidly detect a target nucleic acid with high sensitivity in a point-of-care setting and has become a popular molecular diagnosis method. We show a typical SDA reaction with WarmStart Nt.BstNBI (NEB #R0725) and Bst 2.0 WarmStart® DNA polymerase (NEB #M0538) to detect hBRCA1 target in HeLa genomic DNA in WarmStart Nt.BstNBI. To learn more and to view our strand displacement amplification and nicking enzyme amplification reaction product offerings, please visit here. Q: When is star activity a concern? A: Star activity is of concern if extra banding can cause misinterpretation of results in genotyping and mutational analysis procedures. Experimental design can promote star activity. Small reaction volumes are more likely to contain glycerol concentrations of 5% or greater, a condition known to increase star activity. A 5% glycerol concentration occurs when setting up a double digest in a 20 µl reaction using 1 µl of each enzyme. Overnight digests are more likely to generate star activity. For tips on avoiding star activity, please click here. Q: Which NEB restriction enzymes are supplied with Gel Loading Dye, Purple (6X)? A: All HF-restriction enzymes and most NEB non-HF restriction enzymes are supplied with Gel Loading Dye, Purple (6X). The non-HF restriction enzymes that come supplied with purple dye are: AatII AsiSI BsmBI-v2 EagI MboI NlaIII PvuI SmaI Acil AvrII BspHI EcoRI MboII NotI RsaI SpeI AfeI BamHI BsrGI EcoRV MluI NspI SacI StuI AflII BbsI ClaI Esp3I FseI MseI PacI SacII AgeI BglII DdeI HaeIII MspI PciI SalI XbaI AluI BsaI DpnI HindIII NcoI PmeI SapI XhoI ApaI BseYI DpnII HpaI NdeI PsiI-v2 SfaNI XmaI ApeKI BsiWI DraI KpnI NheI PstI SfiI XmnI AscI * All HF restriction enzymes are also supplied with Gel Loading Dye, Purple (6X) Q: Is Gel Loading Dye, Purple (6X) or Gel Loading Dye, Purple (6X), no SDS compatible with other DNA binding dyes such as SYBR® and GelRed™ during gel electrophoresis? A: Because high affinity nucleic acid binding dyes can affect DNA migration during electrophoresis, post-staining of gels with SYBR or GelRed dyes is highly recommended. However, these dyes can also be used as precast dyes (into the agarose gel). Because the Gel Loading Dye, Purple (6X) has an increased concentration in SDS, some interference may be observed when using SYBR or GelRed as precast dyes. When using these dyes as precast dyes, NEB recommends using our Gel Loading Dye, Purple, No SDS (6X) (NEB #B7025S ) instead. Please follow the recommendations below for both Purple dyes when using SYBR dyes as precast dyes: Reduce the amount of sample DNA loaded to 250 to 500ng, and use 0.5X only of SYBR dyes in precast gels. These dyes are much more sensitive than EtBr. Blown out or smeared bands can be caused by overloading. If using Gel Loading Dye, Purple (6X) B7024S , use TBE instead of TAE buffer, as the SDS interference is increased when using TAE buffer (in the gel and as a running buffer). Use the SYBR dyes as recommended by the manufacturer: add the SYBR dye to the TBE buffer first, then add the SYBR dye/TBE mix to the molten agarose solution. It is preferable to wait a few minutes to add the SYBR dyes to the molten agarose. Adding it to a cooled agarose solution (above gelling temperature, 45°C) produces better results. Always run a freshly made agarose gel, for a single run only. Re-running the gel will yield poor result. Please follow the recommendations below for both Purple dyes when using GelRed dyes as precast dyes: Reduce the amount of sample DNA loaded to 60 to 125ng, and use 0.5X only of GelRed dye in precast gels. This dye is much more sensitive than EtBr. Blown out or smeared bands can be caused by overloading. It is preferable to wait a few minutes to add the GelRed dye to the molten agarose. Adding it to a cooled agarose solution (above gelling temperature, 45°C) produces better results. Always run a freshly made agarose gel, for a single run only. Re-running the gel will yield poor result. SYBR® is a registered trademark of Life Technologies Corporation GelRed™ is a trademark of Biotium