IsoFast® Hot Start Bst Polymerase & Mixes

PB80.42-01 IsoFast Hot Start Bst Mix Product Image

Bst DNA polymerase combined with AptaLock™ reversible hot-start technology power our IsoFast® Hot Start Bst reagents. These products are ideal for all isothermal amplification workflows, giving more sensitive and reliable results faster than ever before.

IsoFast® Hot Start Bst Polymerase is a recombinant version of the large fragment of Geobacillus stearothermophilus (formerly known as Bacillus stearothermophilus, Bst) DNA Polymerase. It is engineered to retain 5’-3’ polymerase activity, but lacks 5’-3’ exonuclease activity. The enzyme’s strong strand displacement capability eliminates the need for denaturation required by standard PCR. Combined with powerful hot start technology, to minimise primer dimer formation and non-specific target amplification, this novel formulation is ideal for non-PCR based amplification

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IsoFast® Hot Start Bst Polymerase & Mix are the most recent and improved development of isothermal amplification technology, designed to deliver unparalleled specificity and speed. Whether you are working with loop-mediated isothermal amplification (LAMP) or other isothermal amplification methods, IsoFast® Hot Start Bst reagents guarantee exceptional sensitivity and reliability. Our reagents harness the power of a hot start Bst DNA polymerase enzyme, which ensures minimal background amplification and maximises target specificity. With IsoFast® Hot Start, your isothermal amplification experiments will be faster, more efficient, and easier than ever in both diagnostic and research applications.

Features

  • AptaLock™ hot start for ultra-sensitive detection of DNA targets
  • Rapid polymerisation for faster time to results (as little as 10 mins)
  • Detect down to 3 target copies per μL
  • Ideal for both cold and room temperature setup
  • Improved speed and sensitivity for early target detection
  • High activity at a broad range of temperatures from 55-70
  • Available with and without fluorescent dye, and as a 2x ready mix
  • Formats available for colourimetric assays.

Applications

  • Whole genome amplification
  • Multiple displacement amplification
  • Rolling circle amplification
  • Isothermal amplification
  • Loop mediated isothermal amplification (LAMP)
  • Molecular diagnostics
  • Point-of-care testing
IsoFast Hot Start Bst vs IsoFast Bst time to result.
Isothermal amplification of a target sequence in the scaffolding protein gene from the M13 bacteriophage genome using IsoFast Hot Start Bst Mix and IsoFast Bst Mix. A primer mix consisting of 0.2 µM for F3 and B3 primers, 1.6 µM for FIP and BIP primers and 0.8 µM for LoopF and LoopB primers was used. The total reaction volume was 25 µL. 8 serial dilutions of M13 ssDNA genome were used, starting with a stock of 0.5 ng/µL and using a dilution factor of 10, corresponding to the number of genome copies indicated in the plot. The reaction was run at 65°C for 100 minutes. A BioRad CFX96 Touch instrument was used to record fluorescence every 10 seconds. The time to threshold indicates the time required to reach the same fluorescent threshold. IsoFast Hot Start Bst Mix shows faster amplification when compared to IsoFast Bst Mix.

Hot start isothermal amplification

Hot start is important in isothermal amplification techniques because it helps to prevent non-specific amplification and false-positive results. Isothermal amplification methods, such as loop-mediated isothermal amplification (LAMP), rely on specific primers and enzymes to amplify a target sequence at a constant temperature, typically between 60°C and 65°C. These techniques are widely used in various applications, including molecular diagnostics and pathogen detection.

Why is hot start crucial in isothermal amplification?

  • Preventing non-specific amplification in Bst polymerase isothermal reactions: In isothermal amplification, it’s essential to ensure that the amplification process only targets the specific sequence of interest. Non-specific amplification can occur when primers bind to unintended regions of the template or when amplification enzymes prematurely initiate amplification. Hot start techniques involve inhibiting the amplification reaction until the reaction reaches the desired temperature. This prevents the formation of non-specific products during the initial stages of the reaction when the temperature is still below the optimal amplification temperature.
  • Enhancing Bst polymerase isothermal amplification specificity: By preventing premature amplification, hot start methods increase the specificity of the isothermal amplification reaction. This means that the reaction is less likely to amplify non-target sequences, reducing the risk of false-positive results.
  • Improving isothermal amplification sensitivity with Bst polymerase: Hot start techniques can also improve the sensitivity of isothermal amplification assays. By minimising non-specific amplification, more of the available reagents are reserved for amplifying the target sequence, increasing the efficiency of the reaction and the detection limit.
IsoFast Hot Start vs NEB Warmstart time to result under cold setup
Isothermal amplification of a target sequence in the scaffolding protein gene from the M13 bacteriophage genome using IsoFast Hot Start Bst Mix and NEB WarmStart LAMP Kit. A primer mix consisting of 0.2 µM for F3 and B3 primers, 1.6 µM for FIP and BIP primers and 0.8 µM for LoopF and LoopB primers was used. The total reaction volume was 25 µL. 8 serial dilutions of M13 ssDNA genome were used, starting with a stock of 0.5 ng/µL and using a dilution factor of 10, corresponding to the number of genome copies indicated in the plot. Reaction master mixes and plates were prepared using cold blocks (Cold Setup), for approximately 20 min. The reaction was run at 65°C for 100 minutes. A BioRad CFX96 Touch instrument was used to record fluorescence every 10 seconds. The time to threshold indicates the time required to reach the same fluorescent threshold. IsoFast Hot Start Bst Mix shows faster amplification when compared to NEB WarmStart LAMP Kit, when reactions are setup at cold temperatures.
IsoFast Hot Start vs NEB Warmstart time to result under ambient setup
Isothermal amplification of a target sequence in the scaffolding protein gene from the M13 bacteriophage genome using IsoFast Hot Start Bst Mix and NEB WarmStart LAMP Kit. A primer mix consisting of 0.2 µM for F3 and B3 primers, 1.6 µM for FIP and BIP primers and 0.8 µM for LoopF and LoopB primers was used. The total reaction volume was 25 µL. 8 serial dilutions of M13 ssDNA genome were used, starting with a stock of 0.5 ng/µL and using a dilution factor of 10, corresponding to the number of genome copies indicated in the plot. Reaction master mixes and plates were prepared at room temperature (Ambient Setup), for approximately 20 min. The reaction was run at 65°C for 100 minutes. A BioRad CFX96 Touch instrument was used to record fluorescence every 10 seconds. The time to threshold indicates the time required to reach the same fluorescent threshold. IsoFast Hot Start Bst Mix shows faster amplification when compared to NEB WarmStart LAMP Kit, when reactions are setup at ambient temperatures.

Specifications

IsoFast® Hot Start Bst Polymerase

Component

1600 Units

8000 Units

IsoFast Hot Start Bst Polymerase 8 U/μL

1 x 200 μL

1 x 1 mL

10x IsoFast Buffer A

1 x 500 μL

2 x 1.25 mL

5x IsoFast Buffer B

1 x 1 mL

3 x 1.7 mL

IsoFast® Hot Start Bst Polymerase with Dye

Component

1600 Units

8000 Units

IsoFast Hot Start Bst Polymerase 8 U/μL

1 x 200 μL

1 x 1 mL

10x IsoFast Buffer A

1 x 500 μL

2 x 1.25 mL

5x IsoFast Buffer B

1 x 1 mL

3 x 1.7 mL

20x Fluorescent Dye

2 x 125 μL

2 x 625 μL

IsoFast® Hot Start Bst Mix

Component

100 Reactions

500 Reactions

2x IsoFast Hot Start Bst Mix

1 x 1.25 mL

5 x 1.25 mL

20x Fluorescent Dye

1 x 125 μL

1 x 625 μL

IsoFast® Hot Start Bst Polymerase

Component

IsoFast Hot Start Bst Polymerase 8 U/μL

10x IsoFast Buffer A

5x IsoFast Buffer B



1600 Units

1 x 200 μL

1 x 500 μL

1 x 1 mL

8000 Units

1 x 1 mL

2 x 1.25 mL

3 x 1.7 mL

IsoFast® Hot Start Bst Polymerase with Dye

Component

IsoFast Hot Start Bst Polymerase 8 U/μL

10x IsoFast Buffer A

5x IsoFast Buffer B

20x Fluorescent Dye



1600 Units

1 x 200 μL

1 x 500 μL

1 x 1 mL

2 x 125 μL

8000 Units

1 x 1 mL

2 x 1.25 mL

3 x 1.7 mL

2 x 625 μL

IsoFast® Hot Start Bst Mix

Component

2x IsoFast Hot Start Bst Mix

20x Fluorescent Dye



100 Reactions

1 x 1.25 mL

1 x 125 μL

500 Reactions

5 x 1.25 mL

1 x 625 μL

Reaction Volume

Storage

25 μL

On arrival, products should be stored between -30 and -20 °C. If stored correctly the kit will retain full until the indicated expiry date.

Reaction Volume

25 μL

Storage

On arrival, products should be stored between -30 and -20 °C. If stored correctly the kit will retain full until the indicated expiry date.

FAQs

Can I use products amplified with IsoFast® Hot Start Bst reagents in downstream applications, such as cloning or sequencing?

Certain types of isothermal amplification strategies lend themselves to downstream applications, e.g. RCA (rolling circle amplification) or WGA (whole genome amplification) for NGS. IsoFast® Hot Start Bst reagents can be used for this type of amplification. However, the most common type of isothermal amplification, LAMP, generates multiple species of DNA products, making it unsuitable for cloning and impractical for NGS applications.

What type of nucleic acid can I amplify with IsoFast® Hot Start Bst reagents?

IsoFast® Hot Start Bst Polymerase, IsoFast® Hot Start Bst Mix, IsoFast® Hot Start Bst Colour Mix, and IsoFast® Hot Start Bst Polymerase Colour are all designed for amplification of DNA and cDNA targets. They are not suitable for direct amplification of RNA targets.

In order to amplify RNA targets, we recommend using our IsoFast® Bst 1-Step Mix, for direct 1-step isothermal amplification. Alternatively, we suggest a 2-step format, where RNA is converted to cDNA using one of our UltraScript® reagents, followed by amplification of cDNA with the users’ preferred IsoFast® Hot Start Bst reagent.

IsoFast® Hot Start Bst Polymerase & Mix are the most recent and improved development of isothermal amplification technology, designed to deliver unparalleled specificity and speed. Whether you are working with loop-mediated isothermal amplification (LAMP) or other isothermal amplification methods, IsoFast® Hot Start Bst reagents guarantee exceptional sensitivity and reliability. Our reagents harness the power of a hot start Bst DNA polymerase enzyme, which ensures minimal background amplification and maximises target specificity. With IsoFast® Hot Start, your isothermal amplification experiments will be faster, more efficient, and easier than ever in both diagnostic and research applications.

Features

  • AptaLock™ hot start for ultra-sensitive detection of DNA targets
  • Rapid polymerisation for faster time to results (as little as 10 mins)
  • Detect down to 3 target copies per μL
  • Ideal for both cold and room temperature setup
  • Improved speed and sensitivity for early target detection
  • High activity at a broad range of temperatures from 55-70
  • Available with and without fluorescent dye, and as a 2x ready mix
  • Formats available for colourimetric assays.

Applications

  • Whole genome amplification
  • Multiple displacement amplification
  • Rolling circle amplification
  • Isothermal amplification
  • Loop mediated isothermal amplification (LAMP)
  • Molecular diagnostics
  • Point-of-care testing
IsoFast Hot Start Bst vs IsoFast Bst time to result.
Isothermal amplification of a target sequence in the scaffolding protein gene from the M13 bacteriophage genome using IsoFast Hot Start Bst Mix and IsoFast Bst Mix. A primer mix consisting of 0.2 µM for F3 and B3 primers, 1.6 µM for FIP and BIP primers and 0.8 µM for LoopF and LoopB primers was used. The total reaction volume was 25 µL. 8 serial dilutions of M13 ssDNA genome were used, starting with a stock of 0.5 ng/µL and using a dilution factor of 10, corresponding to the number of genome copies indicated in the plot. The reaction was run at 65°C for 100 minutes. A BioRad CFX96 Touch instrument was used to record fluorescence every 10 seconds. The time to threshold indicates the time required to reach the same fluorescent threshold. IsoFast Hot Start Bst Mix shows faster amplification when compared to IsoFast Bst Mix.

Hot start isothermal amplification

Hot start is important in isothermal amplification techniques because it helps to prevent non-specific amplification and false-positive results. Isothermal amplification methods, such as loop-mediated isothermal amplification (LAMP), rely on specific primers and enzymes to amplify a target sequence at a constant temperature, typically between 60°C and 65°C. These techniques are widely used in various applications, including molecular diagnostics and pathogen detection.

Why is hot start crucial in isothermal amplification?

  • Preventing non-specific amplification in Bst polymerase isothermal reactions: In isothermal amplification, it’s essential to ensure that the amplification process only targets the specific sequence of interest. Non-specific amplification can occur when primers bind to unintended regions of the template or when amplification enzymes prematurely initiate amplification. Hot start techniques involve inhibiting the amplification reaction until the reaction reaches the desired temperature. This prevents the formation of non-specific products during the initial stages of the reaction when the temperature is still below the optimal amplification temperature.
  • Enhancing Bst polymerase isothermal amplification specificity: By preventing premature amplification, hot start methods increase the specificity of the isothermal amplification reaction. This means that the reaction is less likely to amplify non-target sequences, reducing the risk of false-positive results.
  • Improving isothermal amplification sensitivity with Bst polymerase: Hot start techniques can also improve the sensitivity of isothermal amplification assays. By minimising non-specific amplification, more of the available reagents are reserved for amplifying the target sequence, increasing the efficiency of the reaction and the detection limit.
IsoFast Hot Start vs NEB Warmstart time to result under cold setup
Isothermal amplification of a target sequence in the scaffolding protein gene from the M13 bacteriophage genome using IsoFast Hot Start Bst Mix and NEB WarmStart LAMP Kit. A primer mix consisting of 0.2 µM for F3 and B3 primers, 1.6 µM for FIP and BIP primers and 0.8 µM for LoopF and LoopB primers was used. The total reaction volume was 25 µL. 8 serial dilutions of M13 ssDNA genome were used, starting with a stock of 0.5 ng/µL and using a dilution factor of 10, corresponding to the number of genome copies indicated in the plot. Reaction master mixes and plates were prepared using cold blocks (Cold Setup), for approximately 20 min. The reaction was run at 65°C for 100 minutes. A BioRad CFX96 Touch instrument was used to record fluorescence every 10 seconds. The time to threshold indicates the time required to reach the same fluorescent threshold. IsoFast Hot Start Bst Mix shows faster amplification when compared to NEB WarmStart LAMP Kit, when reactions are setup at cold temperatures.
IsoFast Hot Start vs NEB Warmstart time to result under ambient setup
Isothermal amplification of a target sequence in the scaffolding protein gene from the M13 bacteriophage genome using IsoFast Hot Start Bst Mix and NEB WarmStart LAMP Kit. A primer mix consisting of 0.2 µM for F3 and B3 primers, 1.6 µM for FIP and BIP primers and 0.8 µM for LoopF and LoopB primers was used. The total reaction volume was 25 µL. 8 serial dilutions of M13 ssDNA genome were used, starting with a stock of 0.5 ng/µL and using a dilution factor of 10, corresponding to the number of genome copies indicated in the plot. Reaction master mixes and plates were prepared at room temperature (Ambient Setup), for approximately 20 min. The reaction was run at 65°C for 100 minutes. A BioRad CFX96 Touch instrument was used to record fluorescence every 10 seconds. The time to threshold indicates the time required to reach the same fluorescent threshold. IsoFast Hot Start Bst Mix shows faster amplification when compared to NEB WarmStart LAMP Kit, when reactions are setup at ambient temperatures.

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Specifications

IsoFast® Hot Start Bst Polymerase

Component

1600 Units

8000 Units

IsoFast Hot Start Bst Polymerase 8 U/μL

1 x 200 μL

1 x 1 mL

10x IsoFast Buffer A

1 x 500 μL

2 x 1.25 mL

5x IsoFast Buffer B

1 x 1 mL

3 x 1.7 mL

IsoFast® Hot Start Bst Polymerase with Dye

Component

1600 Units

8000 Units

IsoFast Hot Start Bst Polymerase 8 U/μL

1 x 200 μL

1 x 1 mL

10x IsoFast Buffer A

1 x 500 μL

2 x 1.25 mL

5x IsoFast Buffer B

1 x 1 mL

3 x 1.7 mL

20x Fluorescent Dye

2 x 125 μL

2 x 625 μL

IsoFast® Hot Start Bst Mix

Component

100 Reactions

500 Reactions

2x IsoFast Hot Start Bst Mix

1 x 1.25 mL

5 x 1.25 mL

20x Fluorescent Dye

1 x 125 μL

1 x 625 μL

IsoFast® Hot Start Bst Polymerase

Component

IsoFast Hot Start Bst Polymerase 8 U/μL

10x IsoFast Buffer A

5x IsoFast Buffer B



1600 Units

1 x 200 μL

1 x 500 μL

1 x 1 mL

8000 Units

1 x 1 mL

2 x 1.25 mL

3 x 1.7 mL

IsoFast® Hot Start Bst Polymerase with Dye

Component

IsoFast Hot Start Bst Polymerase 8 U/μL

10x IsoFast Buffer A

5x IsoFast Buffer B

20x Fluorescent Dye



1600 Units

1 x 200 μL

1 x 500 μL

1 x 1 mL

2 x 125 μL

8000 Units

1 x 1 mL

2 x 1.25 mL

3 x 1.7 mL

2 x 625 μL

IsoFast® Hot Start Bst Mix

Component

2x IsoFast Hot Start Bst Mix

20x Fluorescent Dye



100 Reactions

1 x 1.25 mL

1 x 125 μL

500 Reactions

5 x 1.25 mL

1 x 625 μL

Reaction Volume

Storage

25 μL

On arrival, products should be stored between -30 and -20 °C. If stored correctly the kit will retain full until the indicated expiry date.

Reaction Volume

25 μL

Storage

On arrival, products should be stored between -30 and -20 °C. If stored correctly the kit will retain full until the indicated expiry date.

FAQs

Can I use products amplified with IsoFast® Hot Start Bst reagents in downstream applications, such as cloning or sequencing?

Certain types of isothermal amplification strategies lend themselves to downstream applications, e.g. RCA (rolling circle amplification) or WGA (whole genome amplification) for NGS. IsoFast® Hot Start Bst reagents can be used for this type of amplification. However, the most common type of isothermal amplification, LAMP, generates multiple species of DNA products, making it unsuitable for cloning and impractical for NGS applications.

What type of nucleic acid can I amplify with IsoFast® Hot Start Bst reagents?

IsoFast® Hot Start Bst Polymerase, IsoFast® Hot Start Bst Mix, IsoFast® Hot Start Bst Colour Mix, and IsoFast® Hot Start Bst Polymerase Colour are all designed for amplification of DNA and cDNA targets. They are not suitable for direct amplification of RNA targets.

In order to amplify RNA targets, we recommend using our IsoFast® Bst 1-Step Mix, for direct 1-step isothermal amplification. Alternatively, we suggest a 2-step format, where RNA is converted to cDNA using one of our UltraScript® reagents, followed by amplification of cDNA with the users’ preferred IsoFast® Hot Start Bst reagent.