New England Biolabs, M0374L, Bst 3.0® DNA Polymerase

Need assistance designing LAMP primers? Use the Related Categories Isothermal Amplification & Strand Displacement Applications Whole Genome Amplification,, Loop-Mediated Isothermal Amplification,, Isothermal Amplification Specification Unit Definition One unit is defined as the amount of enzyme that will incorporate 25 nmol of dNTP into acid insoluble material in 30 minutes at 65°C. Reaction Conditions 1X Isothermal Amplification Buffer II Pack Incubate at 65°C 1X Isothermal Amplification Buffer II Pack 20 mM Tris-HCl 10 mM (NH4)2SO4 150 mM KCl 2 mM MgSO4 0.1% Tween® 20 (pH 8.8 @ 25°C) Activity in NEBuffers Storage Buffer 100 mM KCl 10 mM Tris-HCl 0.1 mM EDTA 1 mM DTT 0.1% Triton® X-100 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 80°C for 5 minutes 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 3.0 DNA Polymerase be used in other NEBuffers? A: Optimal activity is observed in Isothermal Amplification Buffer II and near optimal performance is observed in Isothermal Amplification Buffer. For other buffers we recommend pH 7.5–10 and monovalent salt 50–250 mM. Activity in other NEB Buffers: NEBuffer1.1: 10% NEBuffer2.1: 100% NEBuffer3.1: 100% CutSmart Buffer: 100% ThermoPol: 75% Isothermal Amplification Buffer: 90% Q: Can Bst 3.0 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), T4 DNA Polymerase (NEB# M0203) and the Quick Blunting™ Kit (NEB #E1201) are the best choices for creating blunt DNA products. Q: Can Bst 3.0 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) and Mung Bean Nuclease (NEB# M0250) are the best choices to remove 3' overhangs. Q: Can Bst 3.0 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 3.0 DNA Polymerase be heat inactivated? A: Yes, heat at 80°C for 5 minutes. Q: Can Bst 3.0 DNA Polymerase be used in applications requiring thermal cycling? A: No, the reaction temperatures are too high for enzyme stability. Q: Can Bst 3.0 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 3.0 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 3.0 DNA Polymerase be diluted? A: Yes, it can be diluted and stored in a buffer containing 100 mM KCl. The preferred buffer is 100 mM KCl, 10 mM Tris-HCl (pH7.5), 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100 and 50% glycerol. Diluent B (NEB# B8002S) can also be used. Q: Does Bst 3.0 DNA polymerase have reverse transcriptase activity? A: Yes. To use for RT applications we recommend elevated magnesium concentrations (4–8 mM). For general RT-LAMP reactions we recommend a dedicated reverse transcriptase (WarmStart RTx, NEB #M0380) in addition to the DNA-dependent DNA polymerase, but when a single-enzyme system is desired Bst 3.0 is the enzyme of choice. Q: Does Bst 3.0 DNA polymerase incorporate dUTP? A: Yes. Bst 3.0 DNA polymerase can be used with a mixture of dUTP and dTTP or with complete replacement of dTTP by dUTP. Use of 100% dUTP may reduce amplification thresholds by 10%. Q: Does Bst 3.0 DNA Polymerase have an active 3'→5' proofreading exonuclease? A: No. If you need a proofreading polymerase we recommend DNA Polymerase I, Large (Klenow) Fragment (NEB# M0210) for lower temperature and strand displacement applications or Q5 DNA polymerase (NEB #M0491) for highest fidelity incorporation and PCR. 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: How active is Bst 3.0 at other temperatures? A: <45 °C: 10–25% 45–55 °C: 75% 55–72 °C: 100% Bst 3.0 can be used up to 75 °C, but we recommend 65–72 °C for optimal performance. Q: How do I reduce non-template amplification (NTC) in LAMP reactions with Bst 3.0? A: The most successful way to eliminate non-template amplification from LAMP reactions is to use a different primer set for the amplicon, and we recommend screening 2–3 sets of LAMP primers for any new target. But if primer design is set and non-template amplification is seen, then the temperature of the Bst 3.0 LAMP reaction can be raised up to 72 °C. This higher temperature generally reduces or eliminates the NTC. A third option is to reduce the concentration of Mg in the reaction. 8 mM Mg is generally best for LAMP performance, but for some targets non-template amplification can be reduced by using 5–6 mM Mg. Q: How do I use Antarctic Thermolabile UDG for carryover prevention in LAMP reactions? A: Carryover contamination prevention requires two parts: incorporation of dUTP by a DNA polymerase during amplicon generation, and excision of those uracils in amplified products and amplicon destruction catalyzed by a UDG. For LAMP, reactions should be run with a ~50% inclusion of dUTP mixed with dTTP (e.g. 1.4 mM dATP, dCTP, dGTP, 0.7 mM dTTP and dUTP) and a Bst DNA polymerase should be used for efficient incorporation of dU without significant inhibition of the reaction. For the subsequent destruction of contaminant products, Antarctic Thermolabile UDG is strongly recommended over the more thermostable E. coli UDG. Include 0.5 μL of Antarctic Thermolabile UDG per 25 μL LAMP reaction, and simply set up and run your LAMP reactions as normal. UDG activity during setup and heating to 65 °C will quickly and efficiently destroy any contaminating products. If more stringent decontamination is required, then 10 minutes at 25 °C can be added to the beginning of the workflow. For simplicity, dUTP and UDG have been included in an updated: WarmStart Colorimetric LAMP 2X Master Mix with UDG and Fluorescent LAMP Kit WarmStart Fluorescent LAMP/RT-LAMP Kit (with UDG). Q: What are the main causes of reaction failure using Bst 3.0 DNA Polymerase? A: Temperatures below 50 °C or over 72 °C can significantly reduce enzyme activity. In addition, failure to use the appropriate buffer containing 50–150 mM KCl, 2–15 mM Mg, or pH outside of the range pH 7.5–10 cause reaction failure. See activity in other buffers above. 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: When I thaw Isothermal Amplification Buffer II I see a lot of white precipitate, is this normal? A: Yes. Due to the high concentration of salts (MgSO4, KCl) in 10X Isothermal Amplification Buffer II, there is commonly precipitation during freeze/thaw cycles. This will easily go back into solution with vortexing, and buffer should be thoroughly vortexed before use. Q: When should Bst 3.0 DNA Polymerase be the enzyme of choice? A: Bst 3.0 DNA Polymerase displays high strand displacement activity. It fills a void between thermophilic and mesophilic polymerases. The temperature optimum of 60–72°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, and Bst 3.0 is the fastest, most robust enzyme for isothermal methods such as LAMP. It displays increased tolerance to inhibitors from diagnostic samples, allowing more consistent reactions with less concern for cleanup. Bst 3.0 has high reverse transcriptase activity up to 72 °C, enabling single-enzyme RT-LAMP reactions and cDNA synthesis through difficult secondary structures. Q: Why would I use Bst 3.0 DNA Polymerase? A: Bst 3.0 DNA Polymerase extends all the improved properties of using Bst 2.0 DNA Polymerase. Bst 3.0 provides the fastest amplification times for diagnostic methods such as LAMP, and is significantly more tolerant of amplification inhibitors and is the recommended enzyme for impure samples or direct detection. If using an RNA target, Bst 3.0 should be used as it has the highest level of reverse transciptase activity and can perform single-enzyme RT-LAMP.