5x HOT FIREPol® Blend Master Mix Ready to Load

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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. 

Ready to load hot-start PCR master mix for more demanding PCR assays

  • increased yield, sensitivity and specificity
  • up to 5x higher fidelity
  • suitable for templates up to 5 kb
  • reduced primer dimer formation
  • load directly on gel after PCR

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Catalog number

04-25-00S25

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Details

Overview

Description

5x HOT FIREPol® Blend Master Mix Ready to Load is a premixed ready-to-use solution containing all reagents required for PCR (except template, primers and water), additional compound needed for direct loading onto agarose gel and two tracking dyes (blue and yellow) that allow to monitor progress during electrophoresis.

HOT FIREPol® Blend Master Mix contains two carefully optimized enzymes – HOT FIREPol® DNA polymerase and a proofreading polymerase. This enzyme blend has both the 5’→ 3’ exonuclease activity as well as the 3’→ 5’ proofreading activity. HOT FIREPol® Blend Master Mix exhibits an increased fidelity (up to five fold) compared to regular Taq polymerase.

In addition, the mix also allows amplification of longer fragments.

Generated PCR products are compatible with blunt-end and T/A cloning procedures.

HOT FIREPol® Blend Master Mix exhibits an increased fidelity (up to five fold) compared to regular Taq polymerase.

Recommendations

We recommend using 5x HOT FIREPol® Blend Master Mix Ready to Load in any PCR application that will be visualized by agarose gel electrophoresis and ethidium bromide staining.

5x HOT FIREPol® Blend Master Mix Ready to Load is not recommended for use in applications where spectro-photometric measurements (absorbance or fluorescence) are necessary because yellow and blue dyes can interfere with these applications.

Properties

Concentration: 5x

Hot-start: yes, initial activation in 12-15 min

Ready to load: yes

Fidelity: 5 x Taq

Cloning Type: T/A cloning and Blunt-end cloning

Amplicon Size: up to 5 kb

Mix Components

HOT FIREPol® DNA polymerase: chemically modified FIREPol® DNA Polymerase enabeling hot-start

Proofreading enzyme: to enhance fidelity

5x Blend Master Mix Buffer with 12. 5 mM MgCl2: 1x PCR solution – 2.5 mM MgCl2

1 mM dNTPs of each 1x PCR solution – 200 µM dATP, 200 µM dCTP, 200 µM dGTP and 200 µM dTTP

Bovine serum albumine (BSA) to enhance PCR reaction

Blue dye: migration equivalent to 3.5-4.5 kb DNA fragment

Yellow dye: migration rate in excess of primers in 1% agarose gel: <35-45 bp

Compound that increases sample density for direct loading

Amplicons of various length from different templates
Related Resources

FAQ

What is the function of BSA in Master Mix?

Bovine serum albumine (BSA) is one of the PCR additives that is used to enhance PCR reaction.

It has been proved that BSA increases PCR yields from low purity templates containing iron chloride, hemin, fulminic acid, humic acid, tannic acid, stool extracts and melanin (Al-Soud and Rådström 2000; Scipioni et al. 2008b; Opel et al. 2010).

Moreover, BSA also improves specificity in amplification of regions with secondary structures. It is also reported to prevent reaction components from sticking to tube walls.

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.

What would be the recommended PCR Master Mix for GC-rich samples?
GC-rich DNA is difficult to amplify, because it forms stable secondary structures and may cause unspecific amplification. Solis BioDyne 5x HOT FIREPol® GC Master Mix works very well with GC-rich samples demonstrating excellent amplification of regions with GC content up to 79%, and can amplify a fragment up to 5 kb. We would suggest to start with the recommended protocol. 5x HOT FIREPol® GC Master Mix is supplied with 100% DMSO and 25mM MgCl2 enabling further optimization of the reaction conditions.
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.

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 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.

Storage & Shipping
Storage

Routine storage: -20 oC

Temporary storage for up to 1 month at room temperature has no detrimental effects on the quality of 5x HOT FIREPol® Blend Master Mix Ready to Load

Shipping

At room temperature