Bst 6.0 DNA Polymerase

Item Number

B744264

• Specifications & Purity: EnzymoPure™, Free of DNA endonuclease and exonuclease.
• Stability And Storage: Stored at -20℃ immediately after use.
• Storage Temp: Store at -20°C
• Shipped In: Ice chest + Ice pads
• Grade: EnzymoPure™

Product Description

Aladdin's Bst 6.0 DNA Polymerase is a homolog of Bst DNA Polymerase, Large fragment , which is derived from Bacillus stearothermophilus (Bst). Compared to the large fragment of Bst DNA Polymerase, Bst 6.0 DNA Polymerase has higher 5'→3' DNA polymerization activity, stronger strand displacement ability, tolerance to dUTP, salt, and non-ionic detergents. This product does not possess 5'→3' and 3'→5' exonuclease activities and can be used in various applications such as loop-mediated isothermal amplification (LAMP), crossing priming amplification (CPA), rolling circle amplification (RCA), and isothermal amplification reactions based on RCA. Its optimal working temperature for isothermal amplification is generally between 50-68℃, typically at 65℃, which is dependent on the primers and template DNA used, and needs to be optimized through experiments.Unit definition

Application：

Loop-mediated isothermal amplification (LAMP), cross-primer amplification (CPA), rolling circle amplification (RCA), helicase isothermal amplification (HDA), and other isothermal amplification of DNA, multiple-displacement amplification (MDA), strand-displaced DNA synthesis, whole-genome amplification (WGA), high GC-content DNA sequencing, rapid sequencing of nanogram-level DNA templates, library preparation for DNA sequencing, etc.Enzyme storage buffer: 10mM Tris-HCl, 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.1% Triton X-100, 50% Glycerol (pH 7.5 at 25℃）10X Bst 6.0 Reaction Buffer: 200mM Tris-HCl, 500mM KCl, 100mM (NH4)2SO4, 20mM MgSO4, 1%Tween-20 (pH 8.8 at 25℃).Denaturation or inactivation: Bst 6.0 DNA Polymerase can be inactivated by heating at 80°C for 20 minutes.

Source：

Recombinantly expressed and purified from E. coli.Purity: Free of DNA endonuclease and exonuclease.Applications: Loop-mediated isothermal amplification (LAMP), cross-primer amplification (CPA), rolling circle amplification (RCA), helicase isothermal amplification (HDA), and other isothermal amplification of DNA, multiple-displacement amplification (MDA), strand-displaced DNA synthesis, whole-genome amplification (WGA), high GC-content DNA sequencing, rapid sequencing of nanogram-level DNA templates, library preparation for DNA sequencing, etc.

Enzyme storage buffer：

10mM Tris-HCl, 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.1% Triton X-100, 50% Glycerol (pH 7.5 at 25℃）10X Bst 6.0 Reaction Buffer: 200mM Tris-HCl, 500mM KCl, 100mM (NH4)2SO4, 20mM MgSO4, 1%Tween-20 (pH 8.8 at 25℃).Denaturation or inactivation: Bst 6.0 DNA Polymerase can be inactivated by heating at 80°C for 20 minutes.

Precautions：

Bst 6.0 DNA Polymerase does not possess 5'-＞3' exonuclease activity.The temperature for isothermal amplification should not exceed 70°C. Otherwise, the enzyme will be inactivated.Bst 6.0 DNA Polymerase cannot be used for thermal cycle sequencing or PCR.Always set up a negative control without template DNA for each isothermal amplification experiment to exclude background amplification.This product should be kept on ice during use, and stored at -20°C immediately after use.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation

Instructions for Use：

1. Primer designFor the design of loop-mediated isothermal amplification primers, please refer to http://primerexplorer.jp/e/ and version V5 is recommended. The manual can be downloaded at http://primerexplorer.jp/e/v5_manual/index.html. Refer to this manual for the preliminary screening of loop-mediated isothermal amplification primers (LAMP), and more suitable primers need to be verified by experiments. LAMP primers consist of 4 or 6 (including Loop) primers. It is recommended to design 6 primers for the experiment.2. Set up LAMP reactions on ice as follows:ReagentVolumeFinal concentrationNuclease-free Water (, ST876)(15.6-x)μl-10X Bst 6.0 Reaction Buffer2.5μl1XMgSO4 (100mM) 1.5μl6mM (8mM total)dNTP (25mM each) (, D7373)1.4μl1.4mM eachFIP/BIP Primers (25X, 40μM)1μl1.6μMF3/B3 Primers (25X, 5μM)1μl0.2μMLoop F/B Primers (25X, 10μM)1μl0.4μMTemplatexμl＞ 10 copies or moreBst 6.0 DNA Polymerase (40U/μl)1μl1600U/mlTotal volume25μl-Note 1: After preparing the reaction, add 1μl of high-concentration SYBR Green I to each tube. After isothermal amplification, centrifuge at 8,000×g for 1 min, positive reactions turn fluorescent green, while negative reactions remain colorless or brown. Even without any indicators, positive reactions can be identified by the turbidity of reactions. Negative reactions are clear and transparent.Note 2: To optimize the reaction, adjust the Mg2+ concentration (4-10mM), the amount of enzyme (0.04-0.32U/μl) or change the reaction temperature (50-68℃).Note 3: If analysis is performed by agarose gel electrophoresis or other methods that require opening of the LAMP reaction vessel, set up auxiliary analysis areas and equipment to avoid contamination.Note 4: In order to ensure the reproducibility of the experiment, it is recommended to add template DNA at the last.Note 5: It is strongly recommended to set a negative control without template to confirm the specificity of amplifications.Note 6: To prevent contamination, the reaction mixture should be prepared on a clean bench.Note 7: The preparation of reagents and template DNA should be performed in different areas from the electrophoresis area to avoid contamination.3. Incubate at 65℃ for 60 minutes.4. Incubate at 80℃ for 20min to terminate the reaction.5. If necessary, examine the reaction products by 2.0% agarose gel electrophoresis. The amplification products should show a ladder-like band pattern.