Some applications of this product may require a license which is not provided by the purchase of this product.
For research use only.
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), an additional compound needed for direct loading onto an agarose gel, and two tracking dyes (blue and yellow) that allow monitoring 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 fivefold) compared to regular Taq polymerase.
In addition, the mix also allows the 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 fivefold) compared to regular Taq polymerase.
We recommend using HOT FIREPol® Blend Master Mix Ready to Load in any PCR application that will be visualized by agarose gel electrophoresis and ethidium bromide staining.
HOT FIREPol® Blend Master Mix Ready to Load is not recommended for use in applications where spectrophotometric measurements (absorbance or fluorescence) are necessary because yellow and blue dyes can interfere with these applications.
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
HOT FIREPol® DNA polymerase: chemically modified FIREPol® DNA Polymerase enabeling hot-start
Proofreading enzyme: to enhance fidelity
5x Blend Master Mix Buffer with 7. 5 mM MgCl2: 1x PCR solution – 1.5 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 albumin (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
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.
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.
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.
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.
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.
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.
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)
Solis BioDyne products should be stored at -20°C.
Shipping and temporary storage for up to 1 month at room temperature (*15−25°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.
Routine storage: -20 oC
Temporary storage for up to 1 month at room temperature has no detrimental effects on the quality of HOT FIREPol® Blend Master Mix Ready to Load
At room temperature
Hot-start Master Mix designed to provide highly specific high-yield amplification of GC-rich templates.
High performance hot-start Master Mix for efficient multiplex reaction - analyse up to 18 targets in one reaction.
Robust and reliable hot-start Master Mix with higher fidelity and longer amplification range for more demanding reactions.
Convenient ready-to-load molecular weight marker to determine the size of dsDNA during gel electrophoresis. Contains 13 discrete bands ranging from 250 bp to 10 000 bp.
Convenient ready-to-load molecular weight marker to determine the size of dsDNA during gel electrophoresis. Contains 13 discrete bands ranging from 100 bp to 3000 bp.
