FAQs IsoFast™ Bst 1-Step Mix – Witec AG

Can IsoFast™ Bst 1-Step Mix be heat inactivated?

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

Can IsoFast™ Bst 1-Step Mix  be used at temperatures other than 65°C?

Yes, the enzyme is active between 57°C and 65°C. We do not recommend using it at temperatures outside this range as the activity will drop.

Can IsoFast™ Bst 1-Step Mix be used for thermal cycle sequencing?

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

Does IsoFast™ Bst Polymerase in IsoFast™ 1-Step Mix 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 the dedicated reverse transcriptase RTase Go as indicated.

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 1-Step Mix supplied with dNTPs?

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

What components are included in the IsoFast™ Bst 1-Step Mix kit?

The kit consists of the 2x IsoFast™ Bst Mix (including the amplification buffer, dNTPs, MgSO4, IsoFast™ Bst polymerase) and of RTase Go at concentrations optimized for reverse transcription isothermal amplification. The kit also contains one tube of 20x fluorescence dye that binds to dsDNA for real-time detection of products. The user only needs to add primers and target RNA.

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.