IsoFast™ Bst Polymerase

IsoFast™ Bst Polymerase is a recombinant form of the large fragment of Bst DNA polymerase, containing 5′-3′ polymerase activity and strand displacement activity.

The enzyme offers fast amplification and strong strand displacement capabilities, making it ideal for nucleic acid amplification methods such as whole genome amplification, multiple displacement amplification and isothermal amplification.

Features

  • Has strand-displacing 5′-3′ polymerase activity
  • Lacks 5′-3′ exonuclease activity
  • DNA synthesis is performed at a constant temperature
  • Operates over a broad temperature range, with an optimum of 65ºC
  • Gives rapid and consistent amplification across a wide range of templates
  • Supplied with an advanced 2-part buffer system for higher yields under difficult conditions
  • 30 minute protocol
  • Flexible formats with and without fluorescent dye
  • Also available as a 2x mix
  • Glycerol-free enzyme

 

CAT No.
Product
Size
Price
QTY
PB80.10-01
IsoFast™ Bst Polymerase
1600 Units
$83.00
PB80.10-08
IsoFast™ Bst Polymerase
8000 Units
$330.00
PB80.11-01
IsoFast™ Bst Polymerase with Dye
1600 Units
$101.00
PB80.11-08
IsoFast™ Bst Polymerase with Dye
8000 Units
$383.00
PB80.12-01
IsoFast™ Bst Mix
100 x 25μL Reactions
$215.00
PB80.12-05
IsoFast™ Bst Mix
500 x 25μL Reactions
$865.00
PB80.30-02
Fluorescent Dye
200 x 25μL Reactions
$35.00
PB80.30-10
Fluorescent Dye
1000 x 25μL Reactions
$104.00

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More Information

IsoFast™ Bst Polymerase is a recombinant protein expressed in E. coli and represents the large fragment of Geobacillus stearothermophilus (formerly known as Bacillus stearothermophilus) DNA Polymerase. This portion of the protein catalyses the 5′-3′ synthesis of DNA and has strand displacement activity but does not contain the 5’-3’ exonuclease domain.1

Strong strand displacement

Strand displacement refers to the ability of an enzyme to dissociate the hydrogen bond of double stranded template DNA encountered downstream, essentially unzipping the DNA as the complementary strand is synthesised. IsoFast™ Bst Polymerase displays strong strand displacement activity and is suitable for amplification methods including whole genome amplification, multiple displacement amplification and isothermal amplification. DNA synthesis is performed at a constant temperature, and we recommend running the reaction at 65ºC. However IsoFast™ Bst Polymerase works well over a broad temperature range (55ºC to 70ºC, see Figure 4), giving a wider range of reaction conditions in order to optimise primer annealing and strand displacement. The enzyme is heat inactivated at 80ºC.

Rapid and consistent results

Designed for fast amplification speed, IsoFast™ Bst Polymerase gives rapid and consistent results across different target sequences (see Figure 1, 2 & 3). The enzyme is provided with an advanced 2-part buffer system to ensure higher yield and performance even under difficult conditions. Amplification can be detected in a number of ways including real-time fluorescence detection and end-point visualisation.

Format flexibility

For added convenience the enzyme is available with separate fluorescent dye (enabling real-time detection with any qPCR thermocycler), and in a 2x mix format for those looking for reduced setup times. The enzyme gives consistent results regardless of the format chosen (see Figure 5).

This product is not suitable for PCR.

Mead DA, McClary JA, Luckey JA, Kostichka AJ, Witney FR, Smith LM. Bst DNA polymerase permits rapid sequence analysis from nanogram amounts of template. Biotechniques. 1991 Jul;11(1):76-8, 80, 82-87.

 

Applications

  • Whole genome amplification
  • Multiple displacement amplification
  • Isothermal amplification
  • Loop mediated isothermal amplification (LAMP)
  • Molecular diagnostics
  • Field diagnostics

Specifications

IsoFast™ Bst Polymerase

Component

1600 Units

8000 Units

IsoFast Bst Polymerase 8U/µL

1 x 200µL

1 x 1mL

10x IsoFast Buffer A

1 x 500µL

2 x 1.25mL

5x IsoFast Buffer B

1 x 1mL

3 x 1.7mL

IsoFast™ Bst Polymerase with Dye

Component

1600 Units

8000 Units

IsoFast Bst Polymerase 8U/µL

1 x 200µL

1 x 1mL

10x IsoFast Buffer A

1 x 500µL

2 x 1.25mL

5x IsoFast Buffer B

1 x 1mL

3 x 1.7mL

20x Fluorescent Dye

2 x 125µL

2 x 625µL

IsoFast™ Bst Mix

Component

100 Reactions

500 Reactions

2x IsoFast Bst Mix

1 x 1.25mL

5 x 1.25mL

20x Fluorescent Dye

1 x 125µL

1 x 625µL

Fluorescent Dye

Component

200 Reactions

1000 Reactions

20x Fluorescent Dye

1 x 125µL

1 x 625µL

IsoFast™ Bst Polymerase

Component

IsoFast Bst Polymerase 8U/µL

10x IsoFast Buffer A

5x IsoFast Buffer B



1600 Units

1 x 200µL

1 x 500µL

1 x 1mL

8000 Units

1 x 1mL

2 x 1.25mL

3 x 1.7mL

IsoFast™ Bst Polymerase with Dye

Component

IsoFast Bst Polymerase 8U/µL

10x IsoFast Buffer A

5x IsoFast Buffer B

20x Fluorescent Dye



1600 Units

1 x 200µL

1 x 500µL

1 x 1mL

2 x 125µL

8000 Units

1 x 1mL

2 x 1.25mL

3 x 1.7mL

2 x 625µL

IsoFast™ Bst Mix

Component

2x IsoFast Bst Mix

20x Fluorescent Dye



100 Reactions

1 x 1.25mL

1 x 125µL

500 Reactions

5 x 1.25mL

1 x 625µL

Fluorescent Dye

Component

20x Fluorescent Dye



200 Reactions

1 x 125µL

1000 Reactions

1 x 625µL

Reaction Volume

Storage

25μL

On arrival, products should be stored between -30 and -15°C. If stored correctly the kit will retain full activity for 12 months.

Reaction Volume

25μL

Storage

On arrival, products should be stored between -30 and -15°C. If stored correctly the kit will retain full activity for 12 months.

FAQs

Can IsoFast™ Bst Polymerase be heat inactivated?

Yes, IsoFast™ Bst Polymerase can be inactivated by heating the tube at >80°C for 10 minutes.

Can IsoFast™ Bst Polymerase be used at temperatures other than 65°C?

Yes, the enzyme is active between 50°C and 70°C, with an optimum between 60°C and 65°C. We do not recommend using it at temperatures greater than 70°C because it becomes heat inactivated.

Can IsoFast™ Bst Polymerase be used for thermal cycle sequencing?

Unfortunately no, as the reaction temperatures are too high for enzyme stability.

Can IsoFast™ Bst Polymerase be used in labelling reactions and partial fill in reactions?

Yes, IsoFast™ Bst Polymerase can work as a valid alternative to the DNA polymerase I, Klenow Fragment for these applications.

Can IsoFast™ Bst Polymerase be used to blunt DNA, fill in 3′ overhangs or remove 5′ overhangs?

After a restriction enzyme reaction, the end parts of a DNA molecule could be left blunt, with a 5′ overhang (3′ recessed end) or a 3’ overhang (5’ recessed end).

IsoFast™ Bst Polymerase shows only 5’ to 3’ polymerase activity but no 5’ to 3’ or 3’ to 5’ exonuclease activity (i.e. the polymerase has no proofreading activity). Therefore, it can only be used to fill in 5’ overhangs (generating a blunt end), but not to remove 3′ overhangs or 5’ overhangs, as it lacks exonuclease activity.

Can IsoFast™ Bst Polymerase incorporate dUTPs?

Yes, but when dUTP replaces more than 50% of dTTP in the reaction the incorporation is significantly inhibited.

Can IsoFast™ Bst Polymerase initiate to amplify at a nick in the double helix?

Yes, it can start strand synthesis at a nick using the fragment at the 3′ end as the primer. And thanks to the strand displacement activity, it can go on until the end of the molecule or until it dissociates from DNA.

Does IsoFast™ Bst Polymerase have reverse transcriptase activity?

Yes, but it is too low for use in reverse transcription applications. For some primer sets and targets IsoFast™ Bst Polymerase may be used as a single-enzyme (RT/DNA polymerase), but we recommend using a dedicated reverse transcriptase as in IsoFast™ Bst 1-Step Mix.

How do I use the fluorescent dye?

The intercalating fluorescent dye is provided at a 20x concentration. 1.25μL per 25μL reaction is recommended when monitoring reactions on most real time qPCR instruments. The FAM channel of common real-time fluorimeters can be used to read the dye.

How fast should I expect a result?

Amplification of any target varies depending on several factors including primer design, template quality and quantity. Generally, kits in the IsoFast™ Bst range will achieve positive results between 5 and 30 minutes.

Is IsoFast™ Bst Polymerase supplied with dNTPs?

Yes. Both the buffer and the mix already contain dNTPs, so there is no need to order dNTPs separately.

When should IsoFast™ Bst Polymerase be the enzyme of choice?

IsoFast™ Bst Polymerase is a DNA polymerase with a very good strand displacement activity. As it has been isolated from the moderate thermophile Bacillus stearothermophilus (recently renamed as Geobacillus stearothermophilus), the temperature optimum of 60-65°C is higher than the one of DNA Polymerase I, Klenow Fragment or the one of phi29 polymerases (37°C), other frequently used strand displacing polymerases.

This is useful in the design of sequencing strategies as well as isothermal amplification technologies. For example, the higher reaction temperature facilitates sequencing through GC rich regions.

More Information

IsoFast™ Bst Polymerase is a recombinant protein expressed in E. coli and represents the large fragment of Geobacillus stearothermophilus (formerly known as Bacillus stearothermophilus) DNA Polymerase. This portion of the protein catalyses the 5′-3′ synthesis of DNA and has strand displacement activity but does not contain the 5’-3’ exonuclease domain.1

Strong strand displacement

Strand displacement refers to the ability of an enzyme to dissociate the hydrogen bond of double stranded template DNA encountered downstream, essentially unzipping the DNA as the complementary strand is synthesised. IsoFast™ Bst Polymerase displays strong strand displacement activity and is suitable for amplification methods including whole genome amplification, multiple displacement amplification and isothermal amplification. DNA synthesis is performed at a constant temperature, and we recommend running the reaction at 65ºC. However IsoFast™ Bst Polymerase works well over a broad temperature range (55ºC to 70ºC, see Figure 4), giving a wider range of reaction conditions in order to optimise primer annealing and strand displacement. The enzyme is heat inactivated at 80ºC.

Rapid and consistent results

Designed for fast amplification speed, IsoFast™ Bst Polymerase gives rapid and consistent results across different target sequences (see Figure 1, 2 & 3). The enzyme is provided with an advanced 2-part buffer system to ensure higher yield and performance even under difficult conditions. Amplification can be detected in a number of ways including real-time fluorescence detection and end-point visualisation.

Format flexibility

For added convenience the enzyme is available with separate fluorescent dye (enabling real-time detection with any qPCR thermocycler), and in a 2x mix format for those looking for reduced setup times. The enzyme gives consistent results regardless of the format chosen (see Figure 5).

This product is not suitable for PCR.

Mead DA, McClary JA, Luckey JA, Kostichka AJ, Witney FR, Smith LM. Bst DNA polymerase permits rapid sequence analysis from nanogram amounts of template. Biotechniques. 1991 Jul;11(1):76-8, 80, 82-87.

 

Applications

  • Whole genome amplification
  • Multiple displacement amplification
  • Isothermal amplification
  • Loop mediated isothermal amplification (LAMP)
  • Molecular diagnostics
  • Field diagnostics

Specifications

IsoFast™ Bst Polymerase

Component

1600 Units

8000 Units

IsoFast Bst Polymerase 8U/µL

1 x 200µL

1 x 1mL

10x IsoFast Buffer A

1 x 500µL

2 x 1.25mL

5x IsoFast Buffer B

1 x 1mL

3 x 1.7mL

IsoFast™ Bst Polymerase with Dye

Component

1600 Units

8000 Units

IsoFast Bst Polymerase 8U/µL

1 x 200µL

1 x 1mL

10x IsoFast Buffer A

1 x 500µL

2 x 1.25mL

5x IsoFast Buffer B

1 x 1mL

3 x 1.7mL

20x Fluorescent Dye

2 x 125µL

2 x 625µL

IsoFast™ Bst Mix

Component

100 Reactions

500 Reactions

2x IsoFast Bst Mix

1 x 1.25mL

5 x 1.25mL

20x Fluorescent Dye

1 x 125µL

1 x 625µL

Fluorescent Dye

Component

200 Reactions

1000 Reactions

20x Fluorescent Dye

1 x 125µL

1 x 625µL

IsoFast™ Bst Polymerase

Component

IsoFast Bst Polymerase 8U/µL

10x IsoFast Buffer A

5x IsoFast Buffer B



1600 Units

1 x 200µL

1 x 500µL

1 x 1mL

8000 Units

1 x 1mL

2 x 1.25mL

3 x 1.7mL

IsoFast™ Bst Polymerase with Dye

Component

IsoFast Bst Polymerase 8U/µL

10x IsoFast Buffer A

5x IsoFast Buffer B

20x Fluorescent Dye



1600 Units

1 x 200µL

1 x 500µL

1 x 1mL

2 x 125µL

8000 Units

1 x 1mL

2 x 1.25mL

3 x 1.7mL

2 x 625µL

IsoFast™ Bst Mix

Component

2x IsoFast Bst Mix

20x Fluorescent Dye



100 Reactions

1 x 1.25mL

1 x 125µL

500 Reactions

5 x 1.25mL

1 x 625µL

Fluorescent Dye

Component

20x Fluorescent Dye



200 Reactions

1 x 125µL

1000 Reactions

1 x 625µL

Reaction Volume

Storage

25μL

On arrival, products should be stored between -30 and -15°C. If stored correctly the kit will retain full activity for 12 months.

Reaction Volume

25μL

Storage

On arrival, products should be stored between -30 and -15°C. If stored correctly the kit will retain full activity for 12 months.

FAQs

Can IsoFast™ Bst Polymerase be heat inactivated?

Yes, IsoFast™ Bst Polymerase can be inactivated by heating the tube at >80°C for 10 minutes.

Can IsoFast™ Bst Polymerase be used at temperatures other than 65°C?

Yes, the enzyme is active between 50°C and 70°C, with an optimum between 60°C and 65°C. We do not recommend using it at temperatures greater than 70°C because it becomes heat inactivated.

Can IsoFast™ Bst Polymerase be used for thermal cycle sequencing?

Unfortunately no, as the reaction temperatures are too high for enzyme stability.

Can IsoFast™ Bst Polymerase be used in labelling reactions and partial fill in reactions?

Yes, IsoFast™ Bst Polymerase can work as a valid alternative to the DNA polymerase I, Klenow Fragment for these applications.

Can IsoFast™ Bst Polymerase be used to blunt DNA, fill in 3′ overhangs or remove 5′ overhangs?

After a restriction enzyme reaction, the end parts of a DNA molecule could be left blunt, with a 5′ overhang (3′ recessed end) or a 3’ overhang (5’ recessed end).

IsoFast™ Bst Polymerase shows only 5’ to 3’ polymerase activity but no 5’ to 3’ or 3’ to 5’ exonuclease activity (i.e. the polymerase has no proofreading activity). Therefore, it can only be used to fill in 5’ overhangs (generating a blunt end), but not to remove 3′ overhangs or 5’ overhangs, as it lacks exonuclease activity.

Can IsoFast™ Bst Polymerase incorporate dUTPs?

Yes, but when dUTP replaces more than 50% of dTTP in the reaction the incorporation is significantly inhibited.

Can IsoFast™ Bst Polymerase initiate to amplify at a nick in the double helix?

Yes, it can start strand synthesis at a nick using the fragment at the 3′ end as the primer. And thanks to the strand displacement activity, it can go on until the end of the molecule or until it dissociates from DNA.

Does IsoFast™ Bst Polymerase have reverse transcriptase activity?

Yes, but it is too low for use in reverse transcription applications. For some primer sets and targets IsoFast™ Bst Polymerase may be used as a single-enzyme (RT/DNA polymerase), but we recommend using a dedicated reverse transcriptase as in IsoFast™ Bst 1-Step Mix.

How do I use the fluorescent dye?

The intercalating fluorescent dye is provided at a 20x concentration. 1.25μL per 25μL reaction is recommended when monitoring reactions on most real time qPCR instruments. The FAM channel of common real-time fluorimeters can be used to read the dye.

How fast should I expect a result?

Amplification of any target varies depending on several factors including primer design, template quality and quantity. Generally, kits in the IsoFast™ Bst range will achieve positive results between 5 and 30 minutes.

Is IsoFast™ Bst Polymerase supplied with dNTPs?

Yes. Both the buffer and the mix already contain dNTPs, so there is no need to order dNTPs separately.

When should IsoFast™ Bst Polymerase be the enzyme of choice?

IsoFast™ Bst Polymerase is a DNA polymerase with a very good strand displacement activity. As it has been isolated from the moderate thermophile Bacillus stearothermophilus (recently renamed as Geobacillus stearothermophilus), the temperature optimum of 60-65°C is higher than the one of DNA Polymerase I, Klenow Fragment or the one of phi29 polymerases (37°C), other frequently used strand displacing polymerases.

This is useful in the design of sequencing strategies as well as isothermal amplification technologies. For example, the higher reaction temperature facilitates sequencing through GC rich regions.