Bst 8.0 DNA Polymerase produced by Aladdin is a homolog of Bst DNA Polymerase, large fragment, namely a homolog of the large fragment of DNA polymerase from Bacillus stearothermophilus (Bst). Compared with the large fragment of Bst DNA Polymerase, it exhibits stronger 5'→3' DNA polymerase activity, stronger strand displacement ability, dUTP tolerance, salt tolerance, and tolerance to nonionic detergents. Bst 8.0 DNA Polymerase has neither 5'→3' nor 3'→5' exonuclease activity and can be used for loop-mediated isothermal amplification (LAMP), crossing priming amplification (CPA), rolling-circle amplification (RCA), and isothermal amplification reactions based on rolling-circle amplification. The temperature for isothermal amplification mediated by Bst 8.0 DNA Polymerase is generally between 50-72 °C, usually 65 °C. The optimal temperature depends on the primers used and the amplification products and needs to be optimized experimentally.
| Source | Recombinant expression in E. coli |
| Appearance | Sterile liquid |
| Storage Buffer | 10 mM Tris-HCl, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 50% Glycerol (pH 7.5 @ 25 °C). |
| Enzyme Concentration | 40 U/μL |
| Purity | No DNA endonuclease or exonuclease activity. |
| Activity Definition | One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 65 °C. |
Components and Description
| Component | B744265-8KU | B744265-40KU |
|---|
| Bst 8.0 DNA Polymerase (40 U/μL) | 200 μL | 1 mL |
| 10 × Bst 8.0 Reaction Buffer | 600 μL | 3 mL |
| 100 mM MgSO₄ | 400 μL | 2 mL |
Applications
Loop-mediated isothermal amplification (LAMP), reverse transcription loop-mediated isothermal amplification (RT-LAMP) (reaction temperature up to 72 °C), crossing priming amplification (CPA), rolling-circle amplification (RCA), helicase-dependent amplification (HDA), and other DNA isothermal amplification methods, multiple displacement amplification (MDA), strand displacement DNA synthesis, whole genome amplification (WGA), sequencing of DNA with high GC content, rapid sequencing of nanogram-level DNA templates, library preparation and sequencing, etc.
Product Advantages
This product not only exhibits considerable DNA polymerase activity, strand displacement ability, dUTP tolerance, and high-temperature resistance (tolerating temperatures up to 72 °C), but also possesses better reverse transcriptase activity and stronger dUTP tolerance, making it suitable for reverse transcription loop-mediated isothermal amplification (RT-LAMP).
Instructions for Use
1. Primer Design.
The preliminary screening of primers for loop-mediated isothermal amplification can refer to this instruction manual. Suitable primers need to be verified and compared through experiments. LAMP primers consist of 4 or 6 primers (including Loop primers). It is recommended to include Loop primers in the design, that is, to design 6 primers for the experiment.
2. Taking LAMP isothermal amplification as an example, set up the reaction system according to the following table.
| Reagent | Volume | Final concentration |
|---|
| Nuclease-free Water | (15.6-x) μL | - |
| 10 × Bst 8.0 Reaction Buffer | 2.5 μL | 1 × |
| MgSO₄ (100 mM) | 1.5 μL | 6 mM (8 mM total) |
| dNTP (25 mM each) | 1.4 μL | 1.4 mM each |
| FIP/BIP Primers (25×, 40 μM) | 1 μL | 1.6 μM |
| F3/B3 Primers (25×, 5 μM) | 1 μL | 0.2 μM |
| Loop F/B Primers (25×, 10 μM) | 1 μL | 0.4 μM |
| DNA or RNA Sample | x μL | > 10 copies or more |
| Bst 8.0 DNA Polymerase (40 U/μL) | 1 μL | 1600 U/mL |
| Total volume | 25 μL | - |
Note 1: After preparing the reaction system, an appropriate amount of high-concentration SYBR Green I (1 μL) can be added to the cap of each reaction tube containing a 25 μL reaction system. After the isothermal amplification reaction is completed, centrifuge at 8000×g for 1 minute. A fluorescent green reaction system indicates a positive result, while a colorless or brown reaction system indicates a negative result. Alternatively, no indicator needs to be added. After the reaction program is completed, an obviously turbid reaction solution indicates a positive result, while a transparent reaction solution indicates a negative result.
Note 2: If reaction optimization is required, the Mg²⁺ concentration (4-10 mM), enzyme amount (0.04-0.32 U/μL), or reaction temperature (50-72 °C) can be adjusted.
Note 3: If analysis is performed by agarose gel electrophoresis or other methods requiring the opening of LAMP reaction containers, please establish a separate analysis area and equipment to avoid contamination.
Note 4: Since the reaction proceeds relatively rapidly, it is recommended to add template DNA last to ensure experimental reproducibility.
Note 5: It is strongly recommended to include a negative control without template to ensure the specificity of amplification.
Note 6: To prevent contamination during reagent preparation, please ensure that operations are performed in a clean bench.
Note 7: Reagent and template DNA preparation should be performed in areas separate from those used for PCR product electrophoresis and other analyses to avoid contamination.
3. Reaction Program: 65 °C for 60 minutes.
4. Inactivation: 80 °C for 5 minutes.
5. If required, electrophoresis analysis can be performed using a 2.0% agarose gel. The appearance of ladder-like bands of amplified products on the electrophoresis gel indicates a positive result, while the absence of ladder-like bands indicates a negative result.
Storage Conditions:
Store at -20 °C, valid for 2 years; transport at ≤0 °C.
Precautions:
(1) Bst 8.0 DNA Polymerase does not possess 5'→3' exonuclease activity. It is recommended that the isothermal amplification reaction temperature should not exceed 72 °C; otherwise, the enzyme will be rapidly inactivated.
(2) Bst 8.0 DNA Polymerase cannot be used for thermal cycling sequencing or PCR.
(3) It is recommended to include a negative control containing no template DNA in each isothermal amplification experiment to exclude background amplification.
(4) The enzyme should be kept in an ice box or on ice during use and immediately stored at -20 °C after use.
(5) This product is intended for scientific research by professionals only. It must not be used for clinical diagnosis or treatment, food or pharmaceutical applications, or stored in ordinary residential premises.
(6) For your safety and health, please wear a laboratory coat and disposable gloves during operation.
