New England Biolabs, M0402L, Bst 2.0 WarmStart® DNA Polymerase (Glycerol-free)

Related Categories Isothermal Amplification & Strand Displacement Applications Loop-Mediated Isothermal Amplification,, Isothermal Amplification,, DNA Amplification, PCR & qPCR 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. Storage Buffer 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 0.1% Triton® X-100 pH 7.1 @ 25°C Heat Inactivation 80°C for 20 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 rAlbumin. 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 2.0® DNA Polymerase be heat inactivated? A: Yes, heat at 80°C for 20 minutes. Q: Can Bst 2.0® DNA Polymerase be used in applications requiring thermal cycling? A: No, the reaction temperatures are too high for enzyme stability. Q: Can Bst 2.0® 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 2.0® DNA Polymerase be used at temperatures other than 65°C? A: Yes, the enzyme has the following activities: 10-15% at 37°C 30-50% at 50°C 100% at 60-70°C 30-50% at 72°C Use of Bst 2.0 is not recommended at temperatures greater than 72°C because it becomes heat-inactivated. Q: Can Bst 2.0 WarmStart® DNA Polymerase (Glycerol-free) at 120,000 U/ml be diluted prior to use? A: Yes, the 120,000 U/ml enzyme can be diluted in 1X Isothermal Amplification Buffer (Lyo-compatible) to 8,000 U/ml prior to use. The 8,000 U/ml dilution is stable at 4°C for at least 7 days. Q: Does Bst 2.0® DNA polymerase incorporate dUTP? A: Yes. Bst 2.0® 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 cause some inhibition of Bst 2.0 reactions (up to 2-fold decrease in amplification threshold time) but robust performance is maintained. Reference: Kuangwen Hsieh, Peter L. Mage, Andrew T. Csordas, Michael Eisenstein, H. Tom Soh. (2014). Simultaneous elimination of carryover contamination and detection of DNA with uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP). Royal Society of Chemistry. PubdMedID: 24577617 Q: Does B1714: 10X Isothermal Amplification Buffer (Lyo-compatible) include any excipients? A: No, 10X Isothermal Amplification Buffer (Lyo-compatible) does not include any excipients for lyophilization. Excipients should be screened and selected by the end user in the application of interest. @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;}@font-face {font-family:Calibri; panose-1:2 15 5 2 2 2 4 3 2 4; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:-469750017 -1040178053 9 0 511 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman",serif; mso-fareast-font-family:"Times New Roman";}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Calibri; mso-fareast-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi; mso-font-kerning:0pt; mso-ligatures:none;}div.WordSection1 {page:WordSection1; 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: How many Freeze/Thaw cycles can Bst 2.0 WarmStart® DNA Polymerase (Glycerol-free) tolerate? A: Bst 2.0 WarmStart® DNA Polymerase (Glycerol-free) was tested up to 30 Freeze/Thaws at 8,000 U/mL and did not show any activity loss in a LAMP reaction. However, repeated freezing and thawing of this product should be avoided. The enzyme (120,000 U/ml) may be stored at 4°C for up to one month. Q: What are Hot Start and WarmStart® polymerases and when would I use them? A: When setting up room temperature reactions off ice When non-specific amplification is observed What does Hot Start/ Warm Start mean? Our Hot Start and WarmStart polymerases utilize aptamers that inhibit enzyme activity at room temperature, which discourages the formation of nonspecific products. The presence of these aptamers does not alter core enzyme function. The distinction between Hot Start and WarmStart is that Hot Start enzymes are thermophilic while WarmStart® enzymes are mesophilic; the thermodynamic range of the aptamers for Hot Start and WarmStart enzymes are largely similar. Aptamers are engineered oligonucleotides that bind to a specific target molecule through non-covalent interactions and include specific nucleobase modifications that can improve inhibition profiles and/or reduce unintended side effects. The benefit of aptamer-based inhibition over other Hot Start technologies (like antibodies) is that they do not require an activation step and bind reversibly in a temperature-dependent manner. NEB offers polymerases with aptamers for routine PCR, high-fidelity PCR, isothermal amplification, and reverse transcription. These aptamers can target different enzymatic functions for different polymerases. Learn More Why is it called "Hot Start?" Like many non-proofreading, Family A DNA polymerases, Taq Polymerase possesses the ability to add bases onto the end of ssDNA in a template-independent manner even at room temperature, and can result in the addition of non-specific bases onto the ends of DNA primers in the reaction, enabling off-target hybridization and reduced overall reaction specificity. In contrast, at higher temperatures, nonspecific binding is reduced as annealing becomes more stringent. Early methods to mitigate undesired activity at low temperature focused on the exclusion of key reaction components until the reaction temperature was increased, which could then be spiked into the mixture, triggering the reaction under a more restrictive, high temperature condition. Read our feature article, Using aptamers to control enzyme activities Hot Start Taq and beyond, to see data comparing product formation for enzymes with and without aptamer-based inhibition of activity. 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 is the difference between buffers B1714: 10X Isothermal Amplification Buffer (Lyo-compatible) and B0537: 10X Isothermal Amplification Buffer? A: The glycerol-free enzyme will be active in both isothermal amplification buffers but the lyo-compatible buffer (NEB #B1714) is optimized for use in lyophilization workflows. Q: What is the Mg2+ concentration in the 10X Isothermal Amplification Buffer (Lyo-compatible)? A: The 10X Isothermal Amplification Buffer (Lyo-compatible) contains 80 mM MgSO4 and thus 8 mM MgSO4 in the 1X final reaction. Q: When should Bst 2.0® DNA Polymerase be the enzyme of choice? A: Bst 2.0 DNA Polymerase displays high strand displacement activity. It fills a void between thermophilic and mesophilic polymerases. The temperature optimum of 60-70°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. Q: Why would I use Bst 2.0 WarmStart® DNA Polymerase? A: Bst 2.0 WarmStart® DNA Polymerase provides all the improved properties of using Bst 2.0 DNA Polymerase, with the additional benefit of room temperature reaction setup capability. DNA polymerases display undesired, non-templated activity at temperatures below set amplification temperatures and this activity can significantly affect performance and reproducibility. The aptamer in Bst 2.0 WarmStart® inhibits DNA polymerase activity below 45 °C and reactions can therefore be set up at room temperature.