Invitrogen™ Lyo-ready Bst DNA Polymerase

Catalog No.	Quantity	Concentration
A56657	1200 U	40 U/μL
A56655	1200 U	6 U/μL
A56656	6000 U	6 U/μL

Catalog No. A56657 Supplier Invitrogen™ Supplier No. A56657

Description

Invitrogen Lyo-ready Bst DNA Polymerase is a proprietary mutant of Bst DNA polymerase, large fragment, with a fast reaction speed, increased sensitivity, and increased tolerance to inhibitors.

Invitrogen Lyo-ready Bst DNA Polymerase is a proprietary mutant of Bst DNA polymerase, large fragment, with a fast reaction speed, increased sensitivity, and increased tolerance to inhibitors. By retaining strong strand-displacement activity, this enzyme helps enable highly efficient isothermal amplifications such as loop-mediated isothermal amplification (LAMP).

Invitrogen Lyo-ready Bst DNA Polymerase is a proprietary mutant of Bst DNA polymerase, large fragment, with a fast reaction speed, increased sensitivity, and increased tolerance to inhibitors. By retaining strong strand-displacement activity, this enzyme helps enable highly efficient isothermal amplifications such as loop-mediated isothermal amplification (LAMP).

Lyo-ready Bst DNA Polymerase possesses 5´→3´ DNA polymerase activity and lacks 5´→3´ exonuclease activity and 3´→5´ exonuclease activity. It is also a glycerol-free enzyme, enabling assay lyophilization and use in microfluidics-based technologies.

Lyo-ready Bst DNA Polymerase features
• Fast target detection in as little as 10 minutes
• Enhanced sensitivity down to 50 copies using optimized reaction buffer in (RT-)LAMP reaction
• Robust amplification, even from inhibitor-containing RNA or DNA samples
• Ability to optimize LAMP or RT-LAMP reactions to fit your needs

Applications
Lyo-ready Bst DNA Polymerase can be used for a wide variety of applications including:
• Loop-mediated isothermal amplification (LAMP)
• Whole genome amplification (WGA)
• Rolling circle amplification (RCA)
• Recombinase polymerase amplification (RPA)
• Helicase-dependent amplification (HDA)
• Exponential amplification reaction (EXPAR)

Notes
1. If performing real-time LAMP detection with SYTO 9 Green Fluorescent Nucleic Acid Stain, dilute the stock to 50 μM with nuclease-free water before every use.
2. SuperScript IV Reverse Transcriptase and RNaseOUT Recombinant Ribonuclease Inhibitor are recommended for reactions containing RNA targets.

Specifications

• Product Type: Stand-alone enzyme
• For Use With (Application): LAMP, RT-LAMP
• Polymerase: Lyo-ready Bst DNA Polymerase
• Quantity: 1200 U
• Content And Storage:

  • Lyo-ready Bst DNA Polymerase (30 μL at 40 U/μL)
  • 10X Bst Reaction Buffer (1 mL)
  • 200 mM MgCl₂ (1 mL)
  • 5 M Betaine (1 mL)
  
  Store at –15 to –25°C.

• No. of Reactions: 200 Reactions
• Optimal Reaction Temperature: 65°C
• Shipping Condition: Dry Ice
• Concentration: 40 U/μL
• Reaction Time: ≤10 minutes

Frequently Asked Questions (FAQs)

Is it possible to combine an (RT-)LAMP reaction with colorimetric detection methods using Lyo-ready Bst DNA polymerase?

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.

How is 1 unit of enzyme activity defined for Lyo-ready Bst DNA Polymerase?

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.

Does Lyo-ready Bst DNA Polymerase incorporate dUTP?

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.

I noticed that my Lyo-ready Bst DNA Polymerase is frozen after storage at -20 degrees C. Is it still good to use?

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.

Do you offer Lyo-ready Bst DNA Polymerase in lyophilized/dry format?

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.

How can I eliminate non-specific amplification for LAMP reaction using Lyo-ready Bst DNA Polymerase?

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.

Do you offer master mixes containing Bst DNA Polymerase for LAMP or RT-LAMP?

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.

Can I use my reverse transcriptase together with Lyo-ready Bst DNA Polymerase for RT-LAMP?

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.

What applications other than loop-mediated isothermal amplification (LAMP) can Lyo-ready Bst DNA Polymerase be used for?

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.

Can Lyo-ready Bst DNA Polymerase be diluted?

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.

What factors can cause reaction failure using Lyo-ready Bst DNA Polymerase?

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.

Can Lyo-ready Bst DNA Polymerase be inactivated by heat?

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.

Does Lyo-ready Bst DNA Polymerase possess reverse transcriptase activity?

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.

What is the temperature range for Lyo-ready Bst DNA Polymerase?

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.

What is the difference between Lyo-ready Bst DNA Polymerase and Bst DNA Polymerase, Large Fragment?

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.