{"product_id":"neb-m9204s","title":"New England Biolabs, M9204S, Bst-XT WarmStart™ DNA Polymerase","description":"Need assistance designing LAMP primers? Use the  \u003cb\u003eRelated Categories\u003c\/b\u003e Isothermal Amplification \u0026amp; Strand Displacement  \u003cb\u003eApplications\u003c\/b\u003e Loop-Mediated Isothermal Amplification,, Isothermal Amplification,, DNA Amplification, PCR \u0026amp; qPCR  \u003cb\u003eSpecification\u003c\/b\u003e \u003cb\u003eStorage Buffer\u003c\/b\u003e 20 mM Tris-HCl 100 mM KCl 0.5 mM TCEP 0.1 mM EDTA 1X stabilizers 50% Glycerol pH 7.5  \u003cb\u003eFAQ\u003c\/b\u003e Q: Can Bst-XT DNA Polymerase be heat inactivated? A: Yes, heat at 80°C for 20 minutes. Q: Can Bst-XT DNA Polymerase be used at temperatures other than 65°C for LAMP reactions? A: Yes, Bst-XT can be used to perform LAMP reactions across the broad temperature range (50°C - 70°C). Optimal performance can vary between amplicons\/primer sets, so screening temperatures may be beneficial. Use of Bst-XT is not recommended at temperatures greater than 72°C. Q: Can Bst-XT DNA Polymerase be diluted? A: Yes. Bst-XT can be diluted just prior to use. When diluting the 120,000U\/mL Bst-XT glycerol-free product using 1X Bst-XT Isothermal Amplification buffer (down to 8,000U\/mL), the diluted Bst-XT can be stored at 4°C for up to 7 days. Q: Can Bst-XT DNA Polymerase be used in end point colorimetric detection? A: Yes, Bst-XT can be used in end point colorimetric detection using non pH-based metal indicator dyes including hydroxynaphthol blue (HNB). We recommend adding 120 µM HNB in 25uL LAMP\/RT-LAMP reaction. When using HNB, the final MgSO4 concentration may be increased up to 8mM to enhance the color contrast between the positive and negative samples. Q: Can Bst-XT DNA Polymerase be used with other NEB buffers? A: Optimal performance will be achieved using the provided Bst-XT Isothermal Amplification Buffer (NEB #B9250) at 1X and 6 mM MgSO4. However, Bst-XT can successfully be used with Isothermal Amplification Buffer (NEB #B0537) and 6mM MgSO4, though a delay in time to detection will likely be observed. We do not recommend using Isothermal Amplification Buffer II (NEB #B0374) or ThermoPol® Buffer (NEB #B9004) with Bst-XT DNA Polymerase as performance is greatly reduced. Q: How can I optimize LAMP\/RT-LAMP reactions for difficult targets when using Bst-XT? A: For challenging and\/or low copy targets, the following reaction conditions can be modified: Reaction time can be increased up to 40 minutes Enzyme concentration can be increased up to 600 U\/ml Final MgSO4 concentration can be optimized (typically 6-8mM) 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 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 \u0026lt;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 is the recommended concentration of Mg+2 to use with Bst-XT in LAMP reactions? A: We recommend using a total of 6 mM MgSO4 in LAMP and RT-LAMP reactions with Bst-XT. The Bst-XT isothermal Amplification Buffer contains 2 mM MgSO4 at 1X, and therefore supplementing with additional 4 mM MgSO4 using the provided 100 mM MgSO4 (NEB #B1003) is recommended. If further optimization is necessary, the final MgSO4 can be increased up to 8 mM total. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835514900649,"sku":"M9204S","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-m9204s","provider":"Iright","version":"1.0","type":"link"}