Fast Taq Template Visualization SYBR Green qPCR Mix
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Instruction manual- Product Description
- Composition
- Precautions
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Product: Fast Taq Template Visualization SYBR Green qPCR Mix
List price: 600
Brand: GOONIE
Item number: 500-100
Specification: 500T
Product Overview
This product is a 2× ready‑to‑use pre‑mix containing SYBR Green I fluorescent dye. The mix includes a blue dye and features a highly efficient, inhibition‑resistant Taq enzyme as its core component. To perform qPCR, simply add primers, template DNA, and water to achieve a working concentration of 1×. In addition, this product is supplied with a yellow 10× dilution buffer; the color change resulting from mixing the two dyes allows you to track pipetting steps and visualize the template, enabling accurate assessment of whether template has been added based on the solution’s color (blue mix + template diluted with yellow buffer → green qPCR reaction mixture).
This product contains a broad‑spectrum reference dye, making it compatible with a wide range of qPCR instruments on the market. No additional reference dye needs to be added to the qPCR reaction mix for inter‑run normalization.
Storage conditions
This product can be stored at −20°C in the dark for 2 years. After thawing, the qPCR Mix remains stable when stored at 4°C in the dark for up to 3 months; repeated freeze–thaw cycles should be avoided whenever possible. This product is shipped with an ice pack.
I. Template Visualization (Optional)
If liquid‑handling tracking is required, add the Dilution Buffer to the template according to the table below before proceeding with the qPCR reaction; if liquid‑handling tracking is not needed, do not use the Dilution Buffer and skip this step.
Template Status Instructions for Use of 10× Dilution Buffer The final concentration of Dilution Buffer in the template Liquid
If the concentration is too high, first dilute the template with ddH2O to an appropriate concentration, then use the template to further dilute the 10× Dilution Buffer to 1×. For example: add 1 μL of 10× Dilution Buffer to 9 μL of template, mix well, and you will obtain the desired solution. 1 ×
Solid
First, dilute the 10× Dilution Buffer to 1× using ddH2O, and then use an appropriate volume of the 1× Dilution Buffer to dissolve the template. 1 ×
II. Perform the reaction according to the parameters listed in the table below (20 μL reaction system).
Reagent Usage amount Final concentration Fast Taq Template Visualization SYBR Green qPCR Mix 10 μL 1× Forward primer (10 μM) 0.4 μL 0.2 μM Reverse primer (10 μM) 0.4 μL 0.2 μM Template X μL Recommended: 10–200 ng/20 μL Nuclease-Free Water Bring up to 20 μL. Note:
1. The recommended concentration range for primers is 0.1–1.0 μM. In general, a final primer concentration of 0.2 μM in the reaction mixture yields good amplification; however, you may adjust this based on your specific amplification goals and desired outcomes.
2. When using Dilution Buffer for pipetting-based tracking, the amount of template added should not exceed 25% of the total reaction volume, nor fall below 10%; in other words, it should be within the range of 10% to 25%. If the template concentration is below 10%, the color development will be faint, compromising the tracking accuracy; if it exceeds 25%, excessive use of Dilution Buffer may interfere with the qPCR reaction.
3. qPCR exhibits extremely high sensitivity, and the accuracy of the template amount added during assay setup significantly impacts the final quantitative results. It is recommended to dilute the template before adding it to the reaction mixture, as this effectively enhances experimental reproducibility. To ensure assay stability, the recommended template input range is 10–200 ng per 20 μL.
4. If the template is a cDNA stock solution, regardless of whether it contains 10× Dilution Buffer, the volume of template added should not exceed 10% of the total reaction volume.
3. Perform the qPCR reaction according to the following procedure.
Two-step method (recommended)
Number of cycles Temperature Time Pre-degeneration 1 95℃ 30 sec Transgender 40 95℃ 10 sec Annealing/Extension (Lighting) 60℃ 30 sec Melting curve 1 Instrument default Instrument default Three-Step Method
Number of cycles Temperature Time Pre-degeneration 1 95℃ 30 sec Transgender 40 95℃ 10 sec Annealing 55~65℃ 10 sec Extension (Daylighting) 72℃ 30 sec Melting curve 1 Instrument default Instrument default Note: Imaging refers to the acquisition of fluorescence signals.
Data analysis
qPCR assays can perform both relative and absolute quantification of the target gene.
Relative quantitative analysis requires the selection of a reference gene. The formula for calculating the relative expression level of the target gene is: 2^-(∆∆Ct). The following data are used as an example to illustrate this.
Simulation calculation.
Based on the Ct values generated by the instrument, calculate the average Ct values for the target gene and the reference gene.
Average Ct of the reference gene Average Ct of the target gene Control group 15 17 Experimental Group 1 15 18 Experimental Group 2 15 16 Calculate the difference between the target Ct and the reference gene Ct for each group: Control group: 17 − 15 = 2; Experimental group 1: 18 − 15 = 3; Experimental group 2: 16 − 15 = 1. The relative expression level of Experimental group 1 compared to the control group is: 2 − (3 − 2) = 0.5, meaning that the target gene expression in Experimental group 1 is 0.5-fold that of the control group.
The relative expression level of Experimental Group 2 compared to the Control Group is: 2^-(1-2) = 2, meaning that the target gene expression in Experimental Group 2 is twice that of the Control Group.
Absolute quantification requires the synthesis of a separate standard curve using a reference gene.
Principles of Primer Design
1. The length of the amplification products should be kept within the range of 80–200 bp whenever possible.
2. Primers should ideally be 17–25 nucleotides in length, with a GC content of 40–60%. The overall distribution of A, T, G, and C should be as even as possible.
3. Use the BLAST function on bioinformatics websites to verify the specificity of the primers.
Frequently Asked Questions and Solutions
1. A signal appears in the blank control NTC.
The reaction mixture was prepared inside a laminar flow hood to prevent aerosol contamination.
The number of cycles should not be excessive; aim to keep it below 40.
Redesign the primers to prevent primer dimers.
2. The melting curve exhibits a bimodal or multimodal pattern.
The primers exhibit poor specificity and need to be redesigned.
Reduce the primer concentration appropriately.
If the cDNA template is contaminated with genomic DNA, the extracted RNA must be treated with DNase.
3. The Ct value is too high.
If the template concentration is low, increase the amount of template; if the template degrades, prepare a fresh template.
Amplification efficiency is low, and the product is excessively long; redesign the primers, aiming to keep the amplicon length within 80–200 bp.
An inhibitor is present in the reaction system; please reconfigure it.
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Product Composition
Component Specifications
500 reactions (20 μl/reaction)
Item number Fast Taq Template Visualization SYBR Green qPCR Mix 5×1 mL 500-100A 10× Dilution Buffer 1×1 mL 500-100B Nuclease-Free Water 1×5 mL 500-100C -
Precautions
1. This product contains a fluorescent dye; during storage and use, it should be kept away from light as much as possible and protected from repeated freeze-thaw cycles.
2. Use only after the Mix has been completely thawed and melted, and before use, invert the container several times to mix thoroughly; do not vortex or shake vigorously to avoid excessive bubble formation.
3. This product contains a broad-spectrum reference dye, making it compatible with a wide range of qPCR instruments on the market. No additional reference dye needs to be added to the qPCR reaction mix for inter‑run normalization.
Keywords
GOONIE
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