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Some applications of this product  may require a license which is not provided by the purchase of this product.

For research use only. 

Hot-start DNA Polymerase with a unique 30-day stability at room temperature for your everyday PCR needs.

A chemically modified hot-start version of the thermostable Taq DNA polymerase FIREPol®. This enzyme is activated only by pre-incubation at 95°C, preventing any unspecific polymerase activity at lower temperatures during reaction set-up.

  • increased specificity and sensitivity
  • reduced primer-dimer formation
  • reaction set-up and shipment without ice

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Catalog number

01-02-0000S

Quantity

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Details

Reagents Provided
Item Pcs. Vial size
HOT FIREPol® DNA Polymerase 1 100 U | 20 µl
10x Solution S 1 0.1 ml
25 mM MgCl2 1 0.5 ml
HOT FIREPol® 10x Buffer B1 1 0.5 ml
HOT FIREPol® 10x Buffer B2 1 0.5 ml
Overview

Description

HOT FIREPol® is a chemically modified FIREPol® DNA Polymerase.

HOT FIREPol® is inactive at room temperature and is activated by a 15 min incubation step at 95°C. This enables hot-start PCR and minimizes mispriming and extension from non-specifically annealed primers and primer-dimers. Recommended for routine applications (fragment up to 5 kb).

Possesses 5′→3′ polymerase and 5′→3′ exonuclease activity, as well as a non-template-dependent terminal transferase activity, but lacks a 3′→5′ exonuclease (proofreading) activity making the generated product suitable for TA-cloning.

The fidelity of HOT FIREPol® is similar to a regular Taq DNA Polymerase (error rate per nucleotide app. 2.5 x10-5).

Properties

Concentration: 5 U/µl

Hot-start: yes, activated by a 15 min incubation step at 95°C

Error rate per nucleotide per cycle is app. 2.5 x 10-5

Accuracy is app. 4 x 104

Estimated half-life at 95ºC is 1.5 hours.

Storage and Dilution Buffer: 50% glycerol (v/v), 20 mM Tris-HCl pH

Source: Purified from an E.coli strain that carries an overproducing plasmid containing a modified gene of Thermus aquaticus DNA Polymerase.

Reagents

HOT FIREPol® DNA Polymerase (5 U/µl) in 20 mM Tris-HCl pH 8.7 at 25ºC, 100 mM KCl, 0.1 mM EDTA, 50% glycerol (v/v), and stabilizers.

HOT FIREPol® 10x Buffer B1 (without Mg2+ and detergent): 0.7 M Tris-HCl, 0.175 M (NH4)2SO4.

HOT FIREPol® 10x Buffer B2 (without Mg2+): 0.7 M Tris-HCl, 0.175 M (NH4)2SO4, 0.2% w/v Tween-20. HOT FIREPol® 10x Buffer B2 contains non-ionic detergent suppressing inhibitory effects of the trace of DNA extraction buffers and enhancing PCR yield and efficiency.

25 mM MgCl2

10x Solution S is an additive that facilitates the amplification of difficult templates (e.g. GC-rich DNA templates). This solution should be used at a defined final concentration (1x, 2x or 3x solution). 10x Solution S is NOT a reaction buffer and should be used ONLY IF non-specific amplification occurs.

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FAQ

Can I use DMSO as a PCR additive in reactions with FIREPol® and HOT FIREPol® DNA polymerases?

Yes, DMSO can be used in reactions with FIREPol® and HOT FIREPol® DNA polymerases and Master Mixes. DMSO is usually recommended for amplifying regions with high GC-content and stable secondary structures. While testing it is recommended to include one sample with additional 2.5% DMSO to test if it improves the results. For further DMSO optimization, the concentration can be raised in 2.5% increments up to 10 % based on following table.

Final DMSO concentration

2.5 %

5 %

7.5 %

10 %

Additional volume of DMSO (in 20 ul PCR reaction)

0.5 µl

1 µl

1.5 µl

2 µl

My DNA sample contains PCR inhibitors, which impairs my PCR results. What can I do for optimization?

An additional cleanup of DNA samples with ethanol precipitation may help to get rid of PCR inhibitors.

Alternatively, decreasing the concentration of DNA template may also help to minimize the amount of inhibitors. In case the additional purification step is not possible, you may consider optimization of the PCR protocol.

The amount of primer can be increased, however, it should not exceed the suggested limits (0.05-1 uM). If your DNA template is contaminated with SDS (common carryover from the DNA extraction process that impairs the activity of Taq polymerase), you may try alternative PCR buffers supplied with Solis BioDyne DNA Polymerases: HOT FIREPol® 10x Reaction Buffer B2 and FIREPol® 10x Reaction Buffer B. These PCR Buffers contain non-ionic detergent Tween-20 that can neutralize the negative effect of SDS.

Which polymerases / master mixes are compatible with a downstream restriction enzyme digest without cleaning up the PCR reaction?

FIREPol® and HOT FIREPol® DNA Polymerases and Master Mixes, as well as SolisFAST® Master Mix are compatible with most restriction enzymes without cleaning up the PCR reaction product.

SolisFAST® Master Mix with UNG contains dUTP instead of dTTP leading to the generation of PCR products containing uracil. Check your restriction enzyme for the ability to digest U-containing DNA amplicons.

Which DNA polymerases / master mixes should I use to obtain PCR product suitable for Sanger sequencing?

Products generated by our FIREPol®, HOT FIREPol® DNA Polymerases and Master Mixes, as well as SolisFast® Master Mixes may be used for Sanger sequencing.

Prior to the sequencing, the products should be purified to remove excess primers and nucleotides (enzymatically with ExoI/SAP or Spin column-based nucleic acid purification).

Products generated with 5x HOT FIREPol® Blend Master Mix should be cleaned up utilising column-based nucleic acid purification method, because this mix contains proofreading enzyme with 3’-5’ exonuclease activity and may degrade your sequencing primer.

Please note that Ready to Load versions of our Master Mixes are not recommended for use in applications where spectro-photometric measurements (absorbance or fluorescence) are necessary because tracking dyes can interfere with these applications.

Can I use Solis BioDyne polymerases / master mixes for colony PCR?

Yes, Solis BioDyne HOT FIREPol® DNA Polymerase and 5x HOT FIREPol® Blend Master Mix are suitable for colony PCR to determine the presence or absence of insert DNA in plasmid constructs directly from bacterial colonies without culturing or plasmid purification steps.

Sometimes, poor quality of the colony material (e.g. presence of contaminants) could inhibit the PCR reaction. It is important to use freshly grown colonies (overnight growth). If possible, include a positive (e.g. purified plasmid DNA with a desired insert) and a negative control (containing all components except template DNA) in your experimental set-up.

Can I use FIREPol® / HOT FIREPol® to amplify DNA fragments with GC content higher than 60%?

GC-rich DNA is difficult to amplify, because it forms stable secondary structures and may cause mispriming and unspecific amplification.

Solis BioDyne FIREPol® / HOT FIREPol® are quite robust on GC-rich regions. We would suggest to start with the recommended protocol. In case you are not satisfied with the result (failed amplification, low yield, unwanted products), you may consider optimization of the reaction conditions.

Solis BioDyne FIREPol® / HOT FIREPol® are supplied with 100 ul vial of the 10x Solution S that facilitates amplification of GC-rich DNA regions. This additive should be used at a defined working concentration (1x, 2x or 3x in the final reaction volume). For example, to get 2x final concentration of Solution S in 20 ul PCR reaction, add 2 ul of 10x Solution S.

After first-strand cDNA synthesis, how much cDNA template should I use for my PCR?

Recommended final amount of cDNA sample in downstream PCR reaction is up to one tenth of the final reaction volume. Overload of cDNA sample may compromise the downstream PCR, because cDNA sample may contain reaction components that may inhibit your PCR reaction.

How much DNA template (genomic or plasmid DNA) is used for a general PCR?
For low complexity templates (i.e. plasmid, lambda, BAC DNA), use 1 ng – 10 ng of DNA per 20 µl reaction. For higher complexity templates (i.e. genomic DNA), use 10 ng – 100 ng of DNA per 20 µl reaction.
Does Solis BioDyne produce high-fidelity DNA polymerase?

It is critical to understand the fidelity requirements of the experiment. For many applications including standard PCR, the user might not need high-fidelity PCR enzymes.

Solis BioDyne doesn’t produce high-fidelity DNA polymerases.

Solis BioDyne FIREPol® and HOT FIREPol® DNA Polymerase fidelity is similar to regular Taq DNA polymerase (error rate per nucleotide ~2.5x10-5).

From our portfolio, the you can find 5x HOT FIREPol® Blend Master Mixes that contain a blend of HOT FIREPol® DNA polymerase and a proofreading polymerase (5x HOT FIREPol® Blend Master Mix and 5x HOT FIREPol® Blend Master MixReady to Load). This enzyme blend has both the 5’-3’ exonuclease activity as well as the 3’-5’ proofreading activity demonstrating increased (up to 5x) fidelity compared to regular Taq.

What is the difference between Solis BioDyne FIREPol® and HOT FIREPol® enzyme?

Due to Solis BioDyne TAG technology (EU Patent EP2501716 and US Patent No 9,321,999), FIREPol® and HOT FIREPol® DNA Polymerases have enhanced stability at room temperature with no activity loss up to 1 month.

Both DNA Polymerases are highly processive, thermostable and robust enzymes with amplification range up to 5 kb. HOT FIREPol® DNA Polymerase is a chemically modified FIREPol® DNA Polymerase activated by a 12-15 min incubation step at 95º C. This prevents misextension from non-specifically annealed primers and primer-dimers.

Enzymes are usually very viscous liquids. What would you recommend for the most accurate pipetting?

Pipetting viscous liquids like glycerol with an air displacement micropipette is difficult, because viscous liquids have a high resistance to flow. We have a few recommendations for accurate pipetting:

  • Hold the pipette vertical, since more liquid gets picked up into the tip when the pipette is held at an angle.
  • Only put the pipette tip in the liquid.
  • Use a very slow aspiration speed, wait for a few seconds until the liquid retightens and then use a very slow dispensing speed.
What is the function of MgCl₂ in a PCR reaction?

The concentration of the MgCl2 in PCR reaction affects the productivity of your PCR reaction. Higher concentrations of MgCl2 may increase productivity of DNA polymerase, but also may decrease specificity causing unexpected products.

Usually, 1.5-2.5 mM final concentration of MgCl2 is optimal for general PCR. However, optimal concentration of MgCl2 depends on template, buffer, DNA and dNTPs. Solis BioDyne produces Master Mixes at different MgCl2 concentration, which allows greater flexibility in reaction optimisation. For your very first experiment with Master Mix, we recommend to use Master Mix with 1,5 - 2 mM MgCl2 (in final 1x PCR Mix)

What are the storage conditions and stability of Solis BioDyne reagents?

Solis BioDyne products should be stored at -20°C.

Shipping and temporary storage for up to 1 month at room temperature (*1525°C) has no detrimental effects on the quality of Solis BioDyne reagents.

Freeze-thaw stability is tested for each product. Most PCR and qPCR products have passed 30 freeze-thaw cycles with no changes in performance. Specific information is found in Storage and Shipping conditions of each product.

When stored and handled under the recommended conditions, full activity of the reagents is retained until the Expiry Date printed on the tube label.

*World Health Organization (2003). Guidelines for the Storage of Essential Medicines and Other Health Commodities.

Publications

Liquid array diagnostics: A novel method for rapid detection of microbial communities in single-tube multiplex reactions

P. Hiseni et al. / Biotechniques / 2019

Genetic diversity and differentiation of introduced red oak (Quercus rubra) in Germany in comparison with reference native North American populations

T. Pettenkofer et al. / Eur. J. For. Res. / 2019

ARID1A facilitates KRAS signaling-regulated enhancer activity in an AP1-dependent manner in colorectal cancer cells

M. Sen et al. / Clin. Epigenetics / 2019
read more
Storage & Shipping
Storage

Routine storage: -20ºC

Temporary storage for up to 1 month at room temperature has no detrimental effects on the quality of HOT FIREPol® DNA Polymerase.

Shipping

At room temperature