Fast Taq SYBR Green qPCR Mix
The Ct values are stable, and the inter-well reproducibility is excellent.
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Instruction manual- Product Description
- Composition
- Precautions
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Product: Fast Taq SYBR Green qPCR Mix
List price: 500
Brand: GOONIE
Item number: 500-102
Specification: 500T
Product Overview
This product contains a modified high-performance hot-start Taq enzyme, a buffer that effectively suppresses non-specific amplification, SYBR Green I fluorescent dye, dNTPs, and other key components. It is a 2× ready-to-use pre-mix; when using, simply add primers, template, and water to achieve a working concentration of 1×, and you can proceed with qPCR reactions.
This product contains a broad‑range reference dye, making it compatible with a wide variety 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 must be stored at −20°C away from light. After reconstitution, the Mix remains stable at 4°C for up to 3 months; during use, minimize repeated freeze–thaw cycles. Transport with ice packs.
Instructions for Use
1. Perform the reaction according to the parameters listed in the table below (20 μL reaction system).
Reagent Usage amount Final concentration qPCR SYBR Green Fast Taq 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 1~2 μL 10~200 ng/20 μL Nuclease-Free Water Bring up to 20 μL. Note: If the template is cDNA stock solution, the amount of template added should not exceed 10% of the total reaction volume.
II. Perform the qPCR reaction according to the following procedure.
Two-step method (recommended)
Number of cycles Temperature Time Pre-denaturation 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-denaturation 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^-(ΔΔCt). The following example uses simulated data to perform the calculation. Based on the Ct values exported by the instrument, compute the average Ct values for both 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 times that of the control group.
The relative expression level of experimental group 2 compared to the control group is: 2^-(1-2) = 2. In other words, the target gene expression in experimental group 2 is twice that of the control group. Absolute quantification requires synthesizing a separate standard curve using a standard reference material specific to the target gene.
Principles of Primer Design
1. The length of the amplified product should be kept within the range of 100–300 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 bases 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. In the blank control, an NTC signal is observed.
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 contains genomic DNA contamination, 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 80–220 bp.
An inhibitor is present in the reaction system; please reconfigure it.
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Product Composition
Component Specification (500T) Item number Fast Taq SYBR Green qPCR Mix 5×1 mL 500-102A Nuclease-Free Water 1×5 mL 500-102B -
Precautions
1. This product contains a fluorescent dye; during storage and use, it should be protected from light as much as possible and subjected to 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‑range reference dye, making it compatible with a variety of qPCR instruments on the market; no additional reference dye needs to be added to the qPCR reaction mix for inter‑reaction normalization.
Keywords
GOONIE
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