Frequently asked questions

qPCR/PCR Kits & Reagents

qPCR/PCR Kits & Reagents

All dry products supplied in glass lyovials are designed to be dissolved in a volume and provide a 5% excess such that the customer can withdraw enough reagent to have a complete 1 mL final reaction volume. For example, if the mix is resuspended to provide a 2X master mix then the volume of water to be added is 525 uL.

Dry MasterMixes are provided with a complete formulation; only nuclease-free water is required to resuspend the mixture as the product is already buffered.

Once dissolved the products may be stored short term in the fridge (2-8°C) for up to 24 hours, or in the freezer (-20°C) for up to 6 months. Up to 16%(v/v) glycerol (molecular grade) may be added to a 2X mixture to help reduce freeze thaw damage.

Dry MasterMixes can withstand transient excursions in temperatures up to 50°C during transport. Our recommended storage temperature for optimum product
performance and longevity is 15-30°C.

Dry MasterMixes can be stored at ambient temperature. No cold supply chain is needed for transportion.

If you are detecting bacterial sequences other than E.coli, the primers will have to exclude any part of the DNA that is shared between E.coli and the bacteria used (in other words, the primers have to be designed only in the part which is unique for the bacteria used).
If detecting E.coli sequences, There are some traces of bacterial DNA in the Taq polymerase and in the UNG (as it is produced in E.coli). You will have to determine the minimum level and subtract it from the positive signal. Everything above this signal should be considered as positive.

There can be an excess of the probe and the positive result is an artifact of this. Using a no amplification control where the Taq polymerase is left out can assess this. A positive result will then suggest an artifact and different reagent concentrations can be used.

The MasterMix may be contaminated with DNA template or PCR product from a previous PCR. Clean working practices should be used to avoid DNA template contamination. To avoid contamination from previous PCRs an UNG step should be introduced providing dUTP has been used in the dNTP mix.
All Eurogentec MasterMixes contain UNG, it can be ordered separately for the Core kits.

Eurogentec is a specialist in the field of Real-Time PCR. Eurogentec offers a design service and can even do complete Real-Time projects.

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As Yakima Yellow™ and VIC are very similar molecules. Yakima Yellow™ Double-Dye Oligos can be detected with VIC settings. As Yakima Yellow™ is normally combined with Eclipse® Dark Quencher, the background correction for TAMRA should be turned off (in plate set up window).

It is possible to combine a FAM-TAMRA with a Yakima Yellow™- Eclipse® Dark Quencher Double-Dye Oligo. However, most Real-Time thermocycler correct for the background fluorescence of TAMRA, incase TAMRA is used as quencher. This means that the fluorescent signal of the FAM-TAMRA probe is measured correctly, but that the Yakima Yellow™-Eclipse® Dark Quencher signal is overcorrected and therefore gives to a lower sensitivity.
Therefore it is recommended to use dark quenchers when performing multiplex Real-Time PCRs, so lost of sensitivity can be reduced to a minimum.

It is dependent on the equipment which dyes can be combined. The combination that works best on most qPCR cyclers is FAM-Eclipse® Dark Quencher with Yakima Yellow™-Eclipse® Dark Quencher. Both are available from Eurogentec.

In most cases FAM is used for the target gene and Yakima Yellow™ for the control gene, when performing a multiplex Real-Time PCR.
Eurogentec does offer endogenous controls with both FAM and Yakima Yellow™.

For most Double-Dye oligos, which have been designed using the Primer Express™ Software, used in combination with the qPCR MasterMix the following cycling conditions can be used:

50 °C 120s UNG hydrolysis step
95 °C 600s inactivation UNG, activation HotGoldStar
95 °C 15s denaturation } x 40
60 °C 60s annealing-elongation } x 40

For allelic discrimination Double-Dye Oligos, Molecular Beacons, Scorpions® and hybridization probes and the most commonly used systems. The Molecular Beacons and Scorpions® have the advantage that they can be monitored at an optimal temperature, to make the distinguishing power as big as possible. The hybridization probes can be used together with melt curves and the Double-Dye Oligos are the easiest to use.

All probe systems can be used for expression profiling. The Double-Dye Oligos are the most user friendly as they are easy to design, require minimal optimization and are easy to use.

Eurogentec offers Double-Dye Oligos that fit each qPCR cycler.

When designing Molecular Beacons, pay attention to the folding of the probe. It might fold into alternate conformations, which are not well quenched. Change the stem or loop sequence, or both to avoid this.

If the salt concentration of the buffer is too low (below 1 mM MgCl2) the probe does also not fold correctly.

Choose Eurogentec's Molecular Beacons and we will help you with the design.

Design assistance

The commonly used quenchers TAMRA, DABCYL and Deep Dark Quencher I (DDQI) are not suitable to quench TAMRA, ROX, Cy® 3 or Cy® 5. For these long-wave emitting dyes Deep Dark Quencher II (DDQII) should be used. This quencher is available from Eurogentec.

SYBR® Green I is an inexpensive, universal dye which binds to all dsDNA. It can be easily used in combination with a simple primer pair to detect PCR products in Real-Time. This dye is mainly very attractive for researchers analysis lots of different genes. However it is important to do a good primer design to avoid primer dimers, which will also be detected by SYBR® Green I.

A probe system is always specific (except Amplifluor™ probes) and therefore does only detect the gene of interest. If you have a difficult to optimize PCR it will not show you any primer dimers or aspecific products.
With a probe system it is also possible to distinguish between similar sequences with small differences like SNPs or mutations.
In general, probe assays need less optimisation than SYBR® Green I assays.

A probe assay and a SYBR® Green I assay can be equally sensitive. In cases of difficult to optimize PCRs the SYBR® Green I assay might be less beneficial as it shows the total fluorescent signal of primer dimers, aspecific product and wanted product.

This is not possible because the buffer for SYBR® Green I kits and the buffer for probe kits are different. The kits for SYBR® Green I applications contain special stabilizers to prevent the SYBR® Green I from degradation and do differ in pH and salt concentration.

A core kit is a 10x kit that contains all components in separate tubes, which gives a maximum in flexibility. The MasterMixes are 2x kits, with all components premixed. These guarantee a high reproducibility and ease of use. In average Core kits give a bit more sensitive results.
Eurogentec offers both types of kits.

Spinning the 96-well plate or tubes will most of the time do, but the results are more reproducible if the plate or tubes are mixed on a magnetic shaker.

In a one step RT qPCR reaction the RT reaction and the qPCR reaction are done in one and the same tube. The buffer is a combination of a RT and a PCR buffer, in which both enzymes do work. The one step RT qPCR reaction is a closed tube assay, so contamination can be avoided. It saves pipeting steps and time and is easy in handling.
In a two step RT qPCR the RT reaction and the qPCR reaction are done in separate tubes. It gives a more flexible way of working in that the cDNA can be used for more than one qPCR reaction and can be archived, so that it eliminates the need to continually isolate RNA.
Further more it allows the use of oligo d(T) and ramdom nonamer primers in the RT step and specific primers in the PCR step. This will increase the specificity and sensitivity of the assay.
Eurogentec offers both one and two step RT qPCR kits.

A one step RT qPCR is recommended when doing HTS (High Throughput Screening), where the experiment should be as easy and straight forward as possible and when doing experiments where all sources of contamination should be excluded.

When performing a RT it is possible to use three different primer types:

Oligo d(T) primers, which bind to the poly A tail of the RNA and then only transcribe RNA. This will avoid contamination with genomic DNA. As the poly A tail is located at the beginning of the gene it will also lead to more full transcripts.
Ramdom nonamers, which bind anywhere in the genome and allow the reverse transcriptase to fill up the gaps, will leads to high yields.
Specific primers, which bind to the gene of interest, and will therefore give specifics products.
The combination of oligo d(T) primers and random nonamers will give the highest yields and the longest transcripts, whereas specific primers transcribe only specific RNA but reduce the yield.
With an One step RT qPCR kit it is only possible to add specific primers, as it should be avoided that oligo d(T) primers and ramdom nonamers participate in the PCR reaction, giving many aspecific products. As a RT reaction is performed at 40-50°C, the primers can bind with mismatches to the RNA and therefore transcribe unwanted sequences, which then also will be amplified in to consecutive PCR, leading to aspecific PCR products. This disadvantage is inherent to the method.

In a Two step kit the oligo d(T) primers and the ramdom nonamers are included in the kit and will give ride to cDNA. Then two specific primers have to be selected and this will amplify the exact sequence.

qPCR kits contain dUTP and UNG to prevent carry over from previous qPCRs.
The kits contain normal dNTPs, mixed with dUTPs. The dUTPs will be incorporated instead of dTTPs during the qPCR. UNG (Uracil-N-glycosylase) is an enzyme, which hydrolyses the release of free uracil from uracil containing DNA. When the qPCR reaction is started with an initial incubation with UNG, no contamination through carry over from previous qPCRs will be seen, as that DNA will be degraded, so it cannot participate anymore in the PCR process.

When performing an assay with UNG, a first step at 50°C during 2min and a second step at 95°C during 10min has to be added before performing a normal qPCR:

50 °C during 2min
95 °C during 10min

94 °C during 15s
55 ºC-65 ºC during 30s
72 ºC during 60s
repeat during 35 cycles

Hold at 50 °C for ever

The fist step is needed to activate the UNG enzyme, to allow it to degrade U containing ds DNA.
The second step at 95 °C will deactivate the UNG and will activate the HotGoldStar.

All kits meant for the use on the ABI Prism™ 5700, 7000, 7700 and 7900 contain a ROX passive reference as these systems require a constant ROX level to be able to calculate the fluorescent signal (ΔRN) correctly.

By designing exon-exon spanning primers you will only amplify cDNA, as the primers do not bind to gDNA. In this way your amount of PCR products is directly related to the initial amount of cDNA, which is again directly related to the initial amount of RNA.

SYBR® Green I is unstable if diluted in a watery solution and also if it is not kept in the dark. Therefore Eurogentec offers kits that contain DMSO to stabilize the SYBR® Green I. Kits should be stored in the dark.

If the primers and Double-Dye Oligo have been designed using Primer Express™ and are used in combination with the qPCR MasterMix or qPCR MasterMix for SYBR® Green I the following matrices can be used to define the correct primer concentration and the correct probe concentration. First define the optimum for the primers and then determine the best concentration for the probe that fits to the primer optimum.

DNA standards are preferred when starting from genomic DNA. RNA standards are preferred when starting from RNA because then the efficiency of the RT reaction can also be measured.

UNG stands for Uracil-N-glycosylase. It is an enzyme, which hydrolyses uracil-glycosidic bonds in DNA containing dUTP, therefore degrading the DNA into small fragments. In this way contamination from previous qPCR reactions can be avoided.

dUTP is not naturally present in DNA and therefore not preferred by the Taq polymerase. The Taq polymerase will incorporate dUTP, but not with the same efficience as normal nucleotides. Therefore the efficiency of the reaction is reduced compared to a reaction in which on dATP, dCTP, dGTP and dTTP are used.

Eurogentec offers kits that contain no dUTPs. However, these kits have to be used very carefully, as there will be no possibility to digest the PCR products with UNG.

A good endogenous control is not up regulated by the effect that needs studying, so the condition, drug, therapy, medium etcetera. It should be expressed in the same range and there should preferably be no similar genes or pseudogenes of this gene.
Cytoskeletal molecules are often a good choice.

qPCR can be done both as endpoint analysis and Real-Time analysis. In endpoint analysis the fluorescence (ΔRn) is measured when the PCR is complete. In Real-Time analysis the fluorescence is measured in each cycle of the PCR. It is only possible to quantify in real time, as the final yield of PCR products is not proportional to the initial amount of DNA, due to limiting factors at the end of the PCR. Therefore endpoint analysis is often used for allelic discrimination and, Real-Time analysis is used for quantification.

The probe design might be bad or the mismatch created is particularly stable. Try redesigning the primers/probe. It may be worth trying to place the primer over the mutation rather than the probe if a design is particularly difficult. A melt curve can help to indicate at which temperature the best discrimination is obtained. An additional detection step can be programmed in the PCR protocol.

Eurogentec offers a design service for qPCR probes.

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Absolute quantitation is a method in which unknown samples are quantitated by comparing them to a standard curve, based on absolute amounts of DNA.

Relative quantitation is a method in which unknown samples are compared to reference samples to determine the increase or decrease of gene expression. The out come is presented as an index, not an absolute amount.

As fluorophores are sensitive to hydrolysis, Eurogentec recommends resuspending qPCR probes in TE 0.01 instead of just water. If the qPCR probes are desolved in an acidic solution the fluorophores can hydrolyze and will give a lower signal to noise ratio.

For long term storage the Takyon™ qPCR MasterMixes and Core Kits should be stored at -15 °C to -25 °C in a constant temperature freezer. When stored under these conditions the reagents are stable during 12 months (MasterMixes) or 24 months (Core Kits).
For short term storage the Takyon™ qPCR MasterMixes and Core Kits can be stored at 4 °C for 6 months.

The Takyon™ Dry MasterMix dTTP can be stored at ambient temperature (15-35°C) up to 18-months. This is valid for unopened bottles (inert gas inside for long term stability)
!!! Do not expose the dried reagent to light as ROX normalisation dye is light sensitive

Probes can be degraded separating the fluorophore from quencher, leading to a high background level of the fluorophore. When aliquoting the oligonucleotides, sterile tubes and tips must be used to avoid contamination with DNases.

The most likely reason is contamination by either free fluorophores or oligonucleotides that contain the fluorophore but not the quencher. The fluorophores can be removed by HPLC.
All Eurogentec probes for Real-Time PCR are HPLC purified, Mass Spectrometry controlled and HPLC controlled.