Lyo-ready Bst DNA Polymerase (40 U/μL), 1200 U - FAQs

Yes, LAMP can be combined with colorimetric detection using calcein. We recommend adding calcein and MnCl₂ to the reaction mixture at final concentrations of 80 µM and 0.25 mM, respectively. Prior to amplification, calcein binds to Mn²⁺ ions, giving the mixture an orange color. During amplification, Mn²⁺ ions form a complex with pyrophosphate, releasing calcein and causing the reaction mixture to change to a green or yellowish-green color, indicating successful amplification.

One unit of enzyme activity is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into a polynucleotide fraction at 65 degrees C in 30 min.

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Lyo-ready Bst DNA Polymerase has the ability to incorporate dUTP during amplicon generation. Use of 100% of dUTP instead of dTTP in the dNTP mix can lead to slower target detection.

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Lyo-ready Bst DNA Polymerase comes in a storage buffer that does not contain glycerol. Product freezing doesn't affect its performance or stability. We do not recommend freeze-thawing Lyo-ready Bst DNA Polymerase for more than 20 cycles. Lyo-ready Bst DNA Polymerase is stable for 24 months when stored at -20 degrees C.

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No. However, Lyo-ready Bst DNA Polymerase comes in a glycerol-free formulation that can be directly air-dried or lyophilized. Dry formulation maintains stability and shows good performance after reconstitution in 10X Bst Reaction Buffer. Read more about lyophilization and air-drying of Lyo-ready Bst DNA Polymerase in the application note “Assessing Lyo-ready Bst DNA Polymerase Stability after Air-drying and Lyophilization” on thermofisher.com/lamp.

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LAMP method is fast and robust, but can often result in non-specific amplification, leading to false positive results. To help ensure reliable results and control non-specific amplification:
- Make sure that the work environment and reagents are clean. We recommend cleaning the workspace regularly to prevent contamination.
- Set up the reactions on ice to avoid non-specific amplification products.
- To enhance specificity in LAMP, primer design and optimization is required. We recommend following the primer design guidelines in the user guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0029128-lyo-ready-Bst-DNA-polymerase_UG.pdf). Ensure that the primer melting temperature is neither too high nor too low. Low temperature may result in non-specific primer binding, and high temperature may inhibit primer binding to the template.
- Other reaction parameters such as reaction time or Bst DNA Polymerase concentration can also be optimized. Depending on the primer design and template concentration, Bst DNA polymerase amount per reaction can be decreased down to 1 U. If end-point detection is used, time can be an important factor for non-specific amplification. Reducing the incubation time by several minutes may help distinguish the NTC from the sample.

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Yes. Bst DNA Polymerase is available in master mix format together with SuperScript IV Reverse Transcriptase. SuperScript IV RT-LAMP Master Mix (Cat. No. A51801, A51802, A51803) comes in a formulation with glycerol and can be used for amplification from DNA and RNA targets.

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We recommend using SuperScript IV Reverse Transcriptase (Cat. No. 18090010, 18090050, 18090200) for the best results in RT-LAMP. All guidelines about SuperScript IV Reverse Transcriptase use in RT-LAMP application can be found in the user guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0029128-lyo-ready-Bst-DNA-polymerase_UG.pdf). Other reverse transcriptases such as SuperScript III Reverse Transcriptase (Cat. No. 18080093) and Maxima H Minus Reverse Transcriptase (Cat. No. EP0751) have also been tested and can be used in RT-LAMP application. We recommend using at least 3 U of SuperScript III Reverse Transcriptase or 1 U of Maxima H Minus Reverse Transcriptase per 25 µL RT-LAMP reaction. The list of Lyo-ready reverse transcriptases can be found here: thermofisher.com/lyo-ready.

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Lyo-ready Bst DNA Polymerase possesses strong strand displacement activity and retains activity in a range of temperatures from 35 to75 degrees C. Lyo-ready Bst DNA Polymerase can be used in other isothermal applications such as RPA, RCA, and WGA. Learn more by going to thermofisher.com/isothermalamplification.

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Yes. Lyo-ready Bst DNA Polymerase (40 U/µl) can be diluted prior to reaction using 1X Bst reaction buffer. To prepare 1X Bst Reaction Buffer, dilute the included 10X Bst Reaction Buffer with nuclease-free water. We recommend using ⋜6 U of Lyo-ready Bst DNA Polymerase in a 25 µL LAMP reaction.

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Temperatures below 35 degrees C or above 75 degrees C can cause a reduction in Lyo-ready Bst DNA Polymerase enzyme activity. Adding suboptimal amounts of Lyo-ready Bst DNA Polymerase per reaction can also lead to the slowdown or failure of amplification. We recommend using ⋜6 U of Lyo-ready Bst DNA Polymerase in a 25 µL LAMP reaction. A Bst concentration that is ⋜0.04 U/µL is not recommended for LAMP and RT-LAMP reactions.

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Yes. We recommend inactivating Lyo-ready Bst DNA Polymerase by heating at 95 degrees C for 5 min. Inactivation is not necessary in real-time (RT)-LAMP detection, where melt curve analysis is not performed. However, we highly recommended inactivating Lyo-ready Bst DNA Polymerase after most amplification reactions due to its fast amplification speed.

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Lyo-ready Bst DNA Polymerase possesses reverse transcriptase activity, but it is not sufficient for amplification from low amounts of RNA template. We recommend adding a separate reverse transcriptase, such as SuperScript IV Reverse Transcriptase (Cat. No. 18090010, 18090050, 18090200) to the RT-LAMP reaction to help ensure high cDNA yield and fast reaction speed.

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Lyo-ready Bst DNA Polymerase can perform DNA amplification in temperatures ranging from 35 to 75 degrees C. At 37 degrees C, Lyo-ready Bst DNA Polymerase has ~50% activity. To help ensure 100% activity of Lyo-ready Bst DNA Polymerase, we recommend using the optimal reaction temperature of 65 degrees C.

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Lyo-ready Bst DNA Polymerase is a proprietary mutant of Bst DNA Polymerase, Large Fragment. Both variants of Bst polymerase possess strand displacement activity that allows highly efficient isothermal amplification such as loop-mediated isothermal amplification (LAMP). Notably, Lyo-ready Bst DNA Polymerase shows fast reaction speed, increased sensitivity and tolerance to inhibitors. Furthermore, it is formulated in a glycerol-free format enabling assay lyophilization and use in microfluidics-based technologies.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.