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IsoFast™ Bst Polymerase 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.