FIREPol^® DNA Polymerase Kit

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.

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.

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.

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 4 ul of 10x Solution S.

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.

Yes, we do offer high-fidelity polymerase. Check out our Solis Hot Start HiFi DNA Polymerase Kit.

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

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.

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.

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.

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.