Bst DNA Polymerase, Large Fragment

Item Number

B744266

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

Product Description

One activity unit is defined as the amount of enzyme that can incorporate 10nmol of dNTPs into acid insoluble material in 30 minutes at 65℃.

Application：

Loop-mediated isothermal amplification (LAMP), helicase isothermal gene amplification (HDA) and other DNA isothermal amplification; multiple displacement amplification (MDA); whole genome amplification (WGA); high GC content DNA sequencing; rapid sequencing of nanogram-level DNA templates; library preparation for sequencing, etc.Definition of activity: One activity unit is defined as the amount of enzyme that can incorporate 10nmol of dNTPs into acid insoluble material in 30 minutes at 65℃.Enzyme storage buffer: 10mM Tris-HCl (pH 7.5), 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.1% Triton X-100, 50% (v/v) glycerol.10X Bst Reaction Buffer: 200mM Tris-HCl, 100mM KCl, 100mM (NH4)2SO4 , 20mM MgSO4 , 1% Triton X-100 , pH8.8 at 25°C.Inactivation or inhibition: Heat at 80℃ for 20min.

Source：

The recombinant large fragment of Bacillus stearothermophilus DNA Polymerase I expressed in E. coli.Purity: Free of DNA endonuclease and exonuclease.

Enzyme storage buffer：

10mM Tris-HCl (pH 7.5), 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.1% Triton X-100, 50% (v/v) glycerol.

Inactivation or inhibition：

Heat at 80℃ for 20min.

Precautions：

The isothermal amplification reaction temperature should not be higher than 70℃ at which the enzyme will be inactivated.Bst DNA Polymerase, Large Fragment cannot be used for thermal cycle sequencing and PCR.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 experiments.2. Taking LAMP isothermal amplification as an example, set up the following reaction.ComponentVolumeFinal concentrationNuclease-free Water(13.5-x)μl-10X Bst Reaction Buffer2.5μl1XMgSO4 (100mM)1.5μl6mM (8mM total)dNTP (10mM each)3.5μl1.4mM eachFIP/BIP Primers (25X, 40μM)1μl1.6μMF3/B3 Primers (25X, 5μM)1μl0.2μMLoopF/B Primers (25X, 10μM)1μl0.4μMTemplatexμl＞ 10 copies or moreBst DNA Polymerase, Large Fragment (8U/μl)1μl320U/mlTotal volume25μl-Note 1: After all the reagents in the table were added, add 1μl of high-concentration SYBR Green I to each tube. After isothermal amplification, centrifuge at 8,000g for 1 min, and the positive reaction will turn fluorescent green, while the negative reaction will remain colorless or brown. Even without any indicators, the 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 60min.4. Incubate at 80℃ for 20min to stop the reaction.5. If necessary, examine the reaction products by 1.5% agarose gel electrophoresis. The amplified product will show as a gradient band.