New England Biolabs, M0275S, Bst DNA Polymerase, Large Fragment

This product is available in a glycerol-free format. Related Categories Isothermal Amplification & Strand Displacement Applications Whole Genome Amplification,, Loop-Mediated Isothermal Amplification,, Isothermal Amplification Specification Unit Definition One unit is defined at the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 65°C. Reaction Conditions 1X ThermoPol® Reaction Buffer Incubate at 65°C 1X ThermoPol® Reaction Buffer 20 mM Tris-HCl 10 mM (NH4)2SO4 10 mM KCl 2 mM MgSO4 0.1% Triton® X-100 (pH 8.8 @ 25°C) Activity in NEBuffers Storage Buffer 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 0.1% Triton® X-100 pH 7.1 @ 25°C Heat Inactivation 80°C for 20 minutes Molecular Weight Theoretical: 67000 daltons 5' - 3' Exonuclease No 3' - 5' Exonuclease No Strand Displacement ++++ Unit Assay Conditions 50 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM MgCl 2 , 30 nM M13mp18 SS DNA, 70 nM M13 sequencing primer (–47) 24 mer, 200 µM dATP, 200 µM dCTP, 200 µM dGTP, 100 µM dTTP including [ 3 H]-dTTP, and 100 µg/ml BSA. FAQ Q: Are NEB DNA Polymerases supplied with dNTPs? A: No, the dNTPs must be ordered separately. They can be ordered as a convenient mix (Deoxynucleotide (dNTP) Solution Mix (NEB# N0447) with a 10 mM concentration of each deoxynucleotide) or as a set of 4 individual tubes (Deoxynucleotide (dNTP) Solution Set (NEB# N0446) with a 100mM concentration of each deoxynucleotide). Q: Can Bst DNA Polymerase be used in other NEBuffers, including rCutSmart? A: Optimal activity is observed in ThermoPol reaction buffer (200% activity). The unit is assayed in a non-optimal buffer taken from the literature. The enzyme exhibits 50% activity in NEBuffers 1 and 3, and 100% activity in buffers 2 and 4. NEBuffers 1-4 must be supplemented with 0.1% Triton X-100 or 100 µg/ml Recombinant Albumin to achieve these values.It can also be used in rCutsmart Buffer. To see its % functional activity in rCutsmart, and that of other DNA modifying enzymes in the cloning workflow, refer to the Activity of DNA Modifying Enzymes in rCutSmart® Buffer chart. Q: Can Bst DNA Polymerase be used to blunt DNA? A: 5' overhangs (3' recessed ends) can be filled in, but 3' overhangs will not be removed because the enzyme lacks an exonuclease activity. DNA Polymerase I, Large (Klenow) Fragment (NEB# M0210) is the enzyme of choice for this application. Q: Can Bst DNA Polymerase be used to fill in 3' overhangs? A: No, DNA polymerases cannot fill in 3' overhangs. To create blunt ends 3' overhangs must be removed. DNA Polymerase I, Large (Klenow) Fragment (NEB# M0210), T4 DNA Polymerase (NEB# M0203) or Mung Bean Nuclease (NEB# M0250) are the best choices to remove 3' overhangs. Q: Can Bst DNA Polymerase be used to remove 5' overhangs? A: No, DNA polymerases cannot remove 5' overhangs. Use Mung Bean Nuclease (NEB# M0250) to remove 5' overhangs. Q: Can Bst DNA Polymerase be used for thermal cycle sequencing? A: No, the reaction temperatures are too high for enzyme stability. Q: Can Bst DNA Polymerase initiate at a nick in the DNA? A: Yes, it can start strand synthesis at a nick using the 3' OH as the primer. Q: Can Bst DNA Polymerase be used in labeling reactions and partial fill in reactions? A: Yes, Klenow Fragment (3'→5' exo-) (NEB# M0212) is also recommended for these applications. Q: Can Bst DNA Polymerase be diluted? A: Yes, it can be diluted and stored in a buffer containing 0.1% Triton X-100 or 100 μg/ml BSA. The preferred buffer is 50 mM KCl, 10 mM Tris-HCl (pH7.5), 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100 and 50% glycerol. Diluent A (NEB# B8001S) can also be used. Q: Can Bst DNA Polymerase be used at temperatures other than 65°C? A: Yes, the enzyme has the following activities: 10-15% at 37°C 30-45% at 50°C 100% at 60-65°C 20% at 70°C Not recommended at temperatures greater than 70°C because it becomes heat-inactivated. Q: Does Bst DNA Polymerase have an active 3'→5' proofreading exonuclease? A: No. Q: Does Bst DNA Polymerase, Large Fragment incorporate dUTP? A: Yes, but with significant inhibition when dUTP replaces >50% of dTTP in the reaction. For reactions with dUTP we recommend a <50:50 mixture of dUTP and dTTP or use of Bst 2.0® DNA polymerase (M0537). Q: Does Bst DNA polymerase have reverse transcriptase activity? A: Yes, but it is typically too low for use in reverse transcription applications. Certain primer sets and targets can be used with Bst as a single-enzyme RT/DNA polymerase, but we recommend using a dedicated reverse transcriptase. If a single polymerase is desired, Bst 2.0® has significantly higher reverse transcriptase activity and should be used in place of Bst DNA polymerase LF. Q: Does NEB have a master mix for LAMP or RT-LAMP reactions? A: Yes. We offer several options to support LAMP/RT-LAMP protocols. The WarmStart® LAMP Kit (DNA & RNA) (NEB #E1700) includes a 50X LAMP fluorescent dye to support fluorescent detection of LAMP/RT-LAMP reactions. The WarmStart Colorimetric LAMP 2X Master Mix (DNA & RNA) (NEB #M1800) includes a pH-based colorimetric indicator for visual detection of LAMP/RT-LAMP reactions (a pink-to-yellow color change signifies amplification). Updated versions of both products include thermolabile UDG and dUTP to reduce the risk of carryover contamination (NEB #E1708 and NEB #M1804, respectively). In addition, the WarmStart Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG) (NEB #M1708) is compatible with different sample input types and supports multiple detection methods, including hydroxynaphthol blue. All LAMP master mixes include a combination of WarmStart RTx Reverse Transcriptase and Bst 2.0 WarmStart DNA Polymerase for fast and robust amplification from both DNA and RNA targets. Each mix requires only user-supplied LAMP primers and target DNA or RNA samples. Q: What is LAMP and RT-LAMP? A: Loop Mediated Isothermal Amplification (LAMP) is an isothermal amplification method designed to detect a target nucleic acid without requiring sophisticated equipment. It uses a stand-displacing DNA polymerase such as a Bst DNA Polymerase and 4-6 primers recognizing 6-8 distinct regions of target DNA for a highly specific amplification reaction. LAMP provides high sensitivity (to fg or <10 copies of target) but with rapid results: reactions can be performed in as little as 5–10 minutes. Reactions can be performed with limited resources, using a water bath for incubation and detection of results by eye, or with real-time measurement and high-throughput instruments. Detection of RNA targets is accomplished by simple addition of a reverse transcriptase to the LAMP reaction, with RT-LAMP performed as a true one-step, isothermal workflow. WarmStart RTx Reverse Transcriptase (NEB #M0380) is a RNA-directed DNA polymerase coupled with a reversibly-bound aptamer that inhibits RTx activity below 40°C, making it particularly well suited for RT-LAMP. To learn more and to view our LAMP product offerings, please visit the LAMP Application Overview Page. Q: What is the difference between Bst DNA Polymerase, Large Fragment, Bst 2.0, Bst 3.0 and Bst-XT DNA Polymerase? A: All four polymerases are moderately thermostable DNA polymerases with strand displacement activity, enabling them to perform isothermal amplification reactions such as LAMP. Bst 2.0 DNA Polymerase is an in silico designed homolog of Bst DNA Polymerase, Large Fragment. It is engineered for improved properties in LAMP reactions, including salt tolerance, thermostability, and dUTP incorporation. Bst 2.0 is notable for its high specificity. Bst 3.0 offers a few improvements to Bst 2.0, most notably faster amplification speed. Bst 3.0 also has improved performance in higher temperature LAMP reactions (up to 72°C), yet for some targets Bst 3.0 does not retain the high specificity of Bst 2.0. Bst-XT combines the most desirable properties of Bst 2.0 and Bst 3.0. It offers the high specificity of Bst 2.0 and the fast amplification speed of Bst 3.0. Bst-XT is also active across a broader temperature range, enabling LAMP reactions between 50-70°C. Bst-XT WarmStart NEB #M9204 Bst 2.0 WarmStart NEB #M0538 Bst 3.0 NEB #M0374 Amplification Speed ★★★★★ ★★★ ★★★★★ Specificity ★★★★★ ★★★★★ ★★ Room temp. set-up? Enabled Enabled Not recommended Optimal LAMP temp 50-70°C 60-70°C 55-72°C Available glycerol-free Yes NEB #M9205 Yes NEB #M0402 Yes NEB #M0443 ★★★★★ = optimal, recommended product for selected application ★★ or ★★★ = will perform selected application ★ = may perform but not recommended Q: What are the main causes of reaction failure using Bst DNA Polymerase? A: Temperatures over 70°C can significantly reduce enzyme activity, or failure to use the appropriate buffer containing either 0.1% Triton X -100 or 100 μg/ml BSA. See Q1 above. Q: When should Bst DNA Polymerase be the enzyme of choice? A: Bst DNA Polymerase is good at strand displacement. It fills a void between thermophilic and mesophilic polymerases. The temperature optimum of 60-65°C is higher than DNA Polymerase I, Large (Klenow) Fragment (NEB #M0210) and lower than Vent® DNA Polymerase (NEB #M0254), two other strand displacing polymerases. This gives researchers a wider range of reaction conditions to optimize strand displacement and primer annealing. This is useful in the design of sequencing strategies as well as isothermal amplification technologies. The elevated reaction temperature facilitates sequencing through GC rich regions. NEB's Selection of Bst DNA Polymerase-based Products for Isothermal DNA Amplification 5′ to 3′ EXO ACTIVITY AMPLIFICATION SPEED ROOM TEMPERATURE SETUP REVERSE TRANSCRIPTASE ACTIVITY INHIBITOR TOLERANCE APPLICATIONS Bst DNA Polymerase, Full Length ** N/A N/A N/A * Nick translation reactions at elevated temperatures Bst DNA Polymerase, Large Fragment N/A * N/A * * General strand-displacement reactions, original polymerase for LAMP and other diagnostic amplifications Bst 2.0 DNA Polymerase N/A ** N/A ** ** Improved LAMP, SDA, and other amplification reactions Bst 2.0 WarmStart DNA Polymerase N/A ** *** ** ** Consistent, room-temperature, and high-throughput amplification assays Bst 3.0 DNA Polymerase N/A *** ** *** *** Fastest, most robust LAMP and RT-LAMP reactions. High reverse transcriptase activity up to 72°C *** Optimal, recommended product for selected application ** Works well for selected application * Will perform selected application, but is not recommended N/A Not applicable to this application