Hieff UNICON™ ColorGPS qPCR Master Mix (Low Rox) is a pre-solution for 2× real-time quantitative PCR amplification, featuring high fluorescence intensity, high sensitivity and specificity, and high amplification yield. The core component, Hieff UNICON™ Taq DNA Polymerase, is hot-started by antibody method, which can effectively inhibit non-specific amplification caused by primer annealing during sample preparation. At the same time, the formula adds factors to enhance the amplification efficiency of the PCR reaction and facilitators to equalize the amplification of genes with different GC contents (30~70%), so that the quantitative PCR can obtain a good linear relationship in a wide range of quantitative regions. The product utilizes the mixed color change reaction of different dyes to monitor the pipetting operation, which effectively reduces the occurrence of pipetting errors.

Features

Pipetting Tracking: Reduce the Risk of Missed/Addition Errors

Excellent Amplification: Good linearity across a wide linear range

High Precision: Can accurately distinguish templates with a 2-fold concentration difference

Good Repeatability: Small inter-well variability in replicate wells, resulting in better repeatability

Stable and Reliable: The reaction system can be stored at room temperature for 24 hours, and the product remains unaffected after 20 freeze-thaw cycles

Applications

Gene Expression Difference Analysis

Absolute Quantitative Analysis

Specifications

Components

Shipping and Storage

This product should be stored at -25~-15℃ for 1 years.

Figures

 Figure1. Reliable results can be obtained within the dynamic range.

Using 2 µL of plasmid templates with a gradient of 10^1-10^7, the related genes were amplified. Hieff UNICON™ ColorGPS master mix can effectively detect a template amount range of 7 orders of magnitude, achieving good linearity within a broad linear range. 

 Figure2. Can accurately distinguish template concentration differences of 2-fold.

Using 2 µL of plasmid diluted in a 2-fold gradient as the template, the related genes were amplified. Hieff UNICON™ ColorGPS master mix can accurately resolve template concentration differences. 

 Figure3. Small inter-well variability is observed, showing better repeatability.

Using dozens of copies of plasmid as the template, amplification was performed with different fluorescent quantitative reagents according to the procedures specified in their respective instruction manuals. The SD values indicate that, compared to other reverse transcription reagents, the new product has smaller differences between replicate wells and better repeatability. 

 Figure4. More flexible, suitable for standard or rapid procedures.

In the standard procedure, the denaturation time and extension time are set to 10 seconds and 30 seconds, respectively, while in the rapid procedure, the denaturation time and extension time are set to 3 seconds and 10 seconds, respectively, to detect the Ct values corresponding to 10 target genes. The results show that the Ct values obtained with the new product in both standard and rapid procedures have a strong correlation, and the new product can achieve good results using the rapid procedure. 

 Figure5. After the reaction system is prepared, it can remain stable at room temperature for 24 hours.

Using different genes from mouse, human 293 cells, and Arabidopsis thaliana as targets, after preparing the reaction system, it was placed in the dark at different temperatures for 24 hours before detection. Compared to immediate processing, the detection difference for different genes does not exceed 0.5 Ct, indicating that stable results can also be obtained with long-duration operations.                

FAQ

Q:How can one select the appropriate fluorescent quantitative reagents based on different instrument models?

A:Please refer to the "YiSheng Biotechnology YiSheng Biotechnology Dye Method Fluorescence Quantitative PCR Mix Selection Table" for details. Click here to learn more and help you choose the appropriate reagents.

Q:How should the reagents be mixed before use?

A:For molecular enzyme reagents, simply invert and mix them together, then perform a gentle centrifugation. It is not recommended to vigorously shake to mix them.

Q:How to use the yellow diluent? Can it be omitted?

A:Refer to the manual. Ultimately, it is necessary to ensure that after being mixed with the template, the volume is exactly 1×. For example, for 20 μL of cDNA stock solution, add 180 μL of sterile ultrapure water to dilute it 10 times, resulting in 200 μL of diluted template. Take 90 μL of the diluted template and add 10 μL of 10× Dilution Buffer, mix well, and then use it. You can choose not to do this, but it will not affect the result.

Q:Do the cDNA templates need to be diluted? And by what factor?

A:You can choose to use the cDNA stock solution or dilute it. Just make sure that the Ct value of the gene you want falls within the range of 15 to 30. Generally, for every 10-fold dilution of cDNA, the Ct value increases by 3.3.

Q:The reaction system has been prepared, but it can't be loaded onto the machine. What should we do?

A:Store in a dark place and the reaction system must not be left open. These two points are essential. It can be placed in a 4℃ refrigerator or at room temperature for 24 hours. Before using, gently shake and centrifuge the sample before loading it onto the instrument. Before loading, carefully check if the tubes or plates have been contaminated.

Q:How to design primers?

A:

When designing primers, it is best to cross introns. The design method can refer to "Enjoyable! A single article to understand qPCR primer design, a must-have skill for experimental experts!" Also, validated primers can be selected. The selection method can refer to "Where to find ready-made qPCR primer sequences?"

Q: The data report on the instrument shows a "BadRox" error. What should I do?

A:The possible reasons are as follows:

①The instrument has not been calibrated for a long time or it is aging. Usually, the instrument needs to be calibrated once a year. It is necessary to have an instrument engineer perform the calibration or replace the aging components.

② A 10 μL reaction system was used. The normal system is a 20 μL system. Using half of the normal system would result in an excessively low total concentration of Rox, increasing the probability of BadRox occurrence. It is recommended to conduct the experiment using the 20 μL system.

Q:Can the qPCR products of this product be run on a gel electrophoresis?

A:The specificity of qPCR products can usually be determined through the melting curve. If it is still necessary to determine the products through gel electrophoresis, the qPCR product can undergo electrophoresis. Electrophoresis requires Loading Buffer, and the product cannot be directly loaded for electrophoresis.

Q:After the amplification process is completed, no amplification curve was observed?

A:①Confirm whether the signal acquisition function is enabled in the program. If a two-step method is used, the signal acquisition should be set during the annealing extension stage.

② Is the length of the product too long? If the length of the product is set to 500bp or more, this situation may occur. It is recommended that during primer design, the length of the product be controlled within the range of 80-300bp.

③ Are the primers inappropriate? If the primers are not designed correctly, they need to be redesigned. If the primers are correctly designed but seem to be degraded, PAGE electrophoresis can be used to check the integrity of the primers.

④ Template degradation: Prepare the template anew and repeat the experiment.

Q:If the NTC shows a CT value, what should we do?

A:The analysis is conducted by observing the melting curve.

The melting curve shows that the melting peak shape of NTC does not overlap with that of the corresponding gene, and the TM value of NTC is smaller than that of the corresponding gene. In this case, the product corresponding to NTC is a primer dimer. If the melting curve peak shape of the corresponding gene is a single peak, the collection of the signal of the related gene will not be affected.

The melting curve shows that the melting peak shape of NTC overlaps with that of the corresponding gene. In this case, the product corresponding to NTC is likely to be the product of the corresponding gene. The system has been contaminated. We need to check one by one whether the water, primers, or reagents have been contaminated. If none of them have been contaminated, it might be aerosol contamination. Based on the obtained amplification data, the ΔCT value is calculated to determine whether the contamination has a significant impact on the system. If ΔCT ≥ 5, it indicates that the contamination has a minor effect and can be ignored.

Q:What should we do when the CT values are shown for NRT?

A:It indicates the presence of genomic DNA contamination. It is recommended to use an RNA extraction kit that removes the genome for RNA extraction or during reverse transcription by adding a step to remove the genome. Alternatively, when designing the primers, a design across introns should be adopted.

Q:What should be done if the repeatability between the holes is poor?

A:①If the CT value is less than 30, but the repeatability is very poor, what should we do?

Improve the accuracy of sample addition. Avoid adding small volumes and reduce the error of addition. This can be achieved by preparing a large sample system, for example, mixing primers, water, and qPCR mix into a large system or using the method of mixing primers and water into a large system. The large system needs to be mixed evenly.

Improve the accuracy of the pipette's aspiration. Pipettes usually need to be calibrated regularly, once a year.

② What if CT is ≥ 30 but the repeatability is very poor?

At this point, due to the low abundance of the effective template, the randomness of collisions between the template and the primers increases, thereby causing a greater difference between the replicons. It is recommended to try adding 2-3 replicons, and then eliminate the data with large differences for data analysis.

Q:What should we do if there are overlapping peaks in the melting curve?

A:The overlapping peaks in the melting curve are located to the left of the main peak. This might be a situation caused by primer dimerization. One possible solution could be to increase the annealing temperature, reduce the primer concentration, or redesign the primers.

The overlapping peaks in the melting curve are located to the right of the main peak.

a. It is possible that the non-specific amplification occurred due to poor specificity of the primers. The primers need to be redesigned.

b. It is possible that the template DNA was contaminated with genomic DNA. It is recommended to use an RNA extraction kit that removes the genomic DNA for RNA extraction, or to add a step of removing the genomic DNA during reverse transcription to reprepare the cDNA template.

Documents:

Manuals

11189_Manual_Ver.EN20260202.pdf