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.
SARS-CoV-2 primers and probes, applicable positive and internal extraction controls are not provided with the Kit and should be supplied by the user.
Yes, it is possible to use this mix for liquid assays. However, if lyophilization is not desired, we recommend using our dedicated master mixes for regular liquid assays. The closest analogue to SolisFAST® Lyo-Ready qPCR Kit with UNG is SolisFAST® Probe qPCR Mix with UNG, catalog number 28-21-0001.
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
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.
Yes, it is possible to opt for your own set of lyophilisation excipients with the SolisFAST® Lyo-compatible qPCR Mix with UNG. However, for the most optimal lyophilisation conditions and in order to achieve the most stable lyophilizates, we highly recommend using the dedicated SolisFAST® Lyo Excipient Mix.
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.
RiboGrip™ is not directly associated with tolerance to common inhibitors, however, it helps to keep RNA intact and prevent RNA degradation because RiboGrip is inactivating ribonuclease enzymes
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.
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:
No. All Solis BioDyne enzymes are genetically modified to enhance their stability at room temperature allowing ice-free reaction set-up.
Dilute your DNA sample with the 6x DNA Loading Dye Buffer: mix 1 volume of the Loading Dye Buffer with 5 volumes of your DNA sample.
Loading Dye Buffers contain glycerol that ensures that the DNA in the ladder and sample forms a layer at the bottom of the well, and color markers (e.g. Bromophenol blue, Xylene cyanole FF, Orange G) for visual tracking of DNA migration during electrophoresis.
For the best results, use Solis BioDyne 6x Loading Dye Buffers (Blue Dye Cat. No. 07-01-00010, Double Blue Dye 07-02-00010, Orange Dye 07-04-00010, Orange Blue Dye 07-03-00010).
All our regular orders are shipped to your doorstep with DHL Express. If You prefer to use different courier, please let us know in comments section (before submitting the order). After receiving an order confirmation, the order will be sent out within one or two working days. Regural shipping fees that apply wordwide (except Estonia) are following:
For a standard electrophoresis system, load 0.4-0.8 µl (0.04-0.08 µg) of Solis BioDyne 100 bp or 1 kb DNA Ladder Ready to Load per 1 mm of gel lane (per 1 mm of well width). It is generally recommended to load the same volumes of your DNA sample and the DNA Ladder.
SolisGreen™ nucleic acid dye shares spectral properties with other commonly used qPCR dyes, allowing detection in the FAM or SYBR® Green I channel (SolisGreen™ excitation maximum 489 nm, emission maximum 515 nm). SolisGreen™ is characterized by high sensitivity and great PCR efficiency with low template amounts for accurate and reproducible results.
SolisGreen™ is based on CYGREEN dye. CYGREEN is used under licence from Enzo Life Sciences, Inc. CYGREEN is a U.S. registered trademark of Enzo Life Sciences, Inc. U.S. Patent Nos. 8,153,802 and 7,569,695.
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.
No. Solis Biodyne 100 bp DNA Ladder and 1 kb DNA Ladder are ready-to-use.
Solis Biodyne 100 bp DNA Ladder and 1 kb DNA Ladder are premixed with 6x DNA Loading Dye Buffer Blue (Cat. No. 07-01-00010) and 6x DNA Loading Dye Buffer Double Blue (Cat. No. 07-02-00010), respectively.
To protect the biological material, such as enzymes, from the temperature and pressure changes during lyophilization, freeze-drying protectants are employed. These protecting excipients ensure that the materials reserve their active state after lyophilisation. In addition to that, they provide an adequate structure to the lyophilized cake or bead.
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.
The 6X Loading Dye buffer is used to prepare DNA ladders and samples for loading on agarose or polyacrylamide gels.
Lyophilization, commonly known as freeze-drying, is a process employed to remove the water content from a substance by sublimation after it has been frozen, while preserving its structure and properties. The reagents can be lyophilized to a spheric lyo-bead or a compact lyo-cake format.
Lyophilization helps to extend the product’s shelf life, enhance the stability of the reagents at higher tempereatures, even when mixed together with primers and probes, and allows for convenient and accurate dispensing of the reagents. After lyophilization, the reagents are easily reconstituted with sample material.
Dye-based detection (e.g., EvaGreen®, SolisGreen®, SYBR® Green) is a cost-effective qPCR option, as it requires only addition of PCR primers. However, the intercalating dye will detect any dsDNA (non-specific amplicons, primer dimers) produced in the reaction. Melt curve analysis performed after the qPCR can be used to verify the specificity of amplification and to check for the presence of non-specific amplification products.
The probe-based qPCR system demonstrates higher specificity compared to dye-based qPCR, because probes only detect the gene of interest. Keep in mind that in probe-based assays, primer dimers and non-specific products will not be detected, however, they may compromise the PCR efficiency. Using probe-based qPCR system, it is possible to distinguish between sequences with high similarity (e.g. single-nucleotide variations). Additionally, probe-based qPCR assays allow for multiplex reactions in one tube, while only a single target can be amplified and measured in a dye-based qPCR.
Both dyes are DNA intercalating agents that are used to stain dsDNA. SolisGreen™ dye is spectrally similar to EvaGreen® allowing detection in the FAM or SYBR® Green I channel (SolisGreen™ excitation maximum 489 nm, emission maximum 515 nm). It has been shown that qPCR mixes based on SolisGreen™ dye have increased performance with low target concentration demonstrating improved precision and less variance between technical replicates.
Both dyes are DNA intercalating agents that are used to stain dsDNA.
EvaGreen® dye is spectrally similar to FAM or SYBR® Green I, which means that no change in optical settings is required for using an EvaGreen®-based qPCR mix.
EvaGreen® was reported to have several benefits over SYBR® Green I:
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.
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.
A molecular-weight size marker used to identify the approximate size (usually in base pairs) and amount (usually in nanograms) of a DNA molecule run on a gel during electrophoresis, using the principle that molecular weight is inversely proportional to migration rate through a gel.
DNA ladder contains double-stranded DNA fragments whose molecule weights (and size in base pairs) are known and standardized. When run alongside your PCR product in a gel, the DNA ladder allows you to estimate the size and amount of your DNA fragment by comparing it to the closest band in the ladder lane.
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)
The concentration of agarose in a gel depends on the sizes of the DNA fragments, and should be able to separate your fragment(s) enough so that they are identifiable.
Smaller fragments of DNA migrate through the gel more quickly and, therefore, are separated on higher concentrations of agarose. Larger molecules require a lower concentration of agarose.
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.
Please click HERE to open the Data Sheet of SolisFAST® Lyo-Ready qPCR Kit with UNG or find it under Related Resources. The Data Sheet includes the recommended lyophilization protocol.
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.
The Kit has been tested and validated by diagnostic laboratories using primer-probe panels recommended by Charité, (Germany) and CDC (USA) protocols according to WHO “Coronavirus disease (COVID-19) technical guidance: Laboratory testing for 2019-nCoV in humans”, section: “Molecular assays to diagnose COVID-19”; subsection: “In-house developed molecular assays”
ROX (carboxy-X-rhodamine) is one of the passive reference dyes providing a constant fluorescent signal that is used for signal normalization during the amplification cycles. The emission recorded from a reference dye during the baseline cycles is used to normalize the emission recorded from the reporter (e.g. EvaGreen®, SolisGreen®, SYBR® Green, etc.) in later cycles in ROX-dependent real-time PCR systems (e.g. Applied Biosystems 5700, 7000, 7300, 7700, 7900HT, StepOne™, StepOnePlus™). Reference dye compensates for small fluorescent fluctuations and well-to-well variations that may occur.
In Solis BioDyne qPCR mixes we use technology, based on ROX, that allows us to use same mix for high- and low- ROX requiering cyclers.
Please check cycler-mix compatibility here if not sure.
