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Cat. No.: SGQRU-100 (for 100T)

Cat. No.: SGQRU-500 (for 500T)

Description

SYBR Green One-Step qRT-PCR Kit (20X, UDG) is a high-quality, carryover prevention premix reagent kit for one-step quantitative reverse transcription PCR (qRT-PCR or real-time RT-PCR) based on SYBR Green dye. It is primarily designed for ultra-sensitive and specific quantification of RNA. This product contains an optimized ratio of high-quality UDG enzyme and dUTP, effectively eliminating the risk of false positives or low CT values caused by product contamination in PCR amplification. The SYBR Green method is often referred to as the dye method, hence this approach is commonly known as the carryover prevention dye method.

UDG (Uracil-DNA Glycosylase), also known as UNG (Uracil-N-glycosylase), catalyzes the hydrolysis of the N-glycosidic bond between the uracil (dU) base in the DNA chain and deoxyribose, releasing free uracil. It is mainly used to eliminate the product contamination issues in PCR amplification. The principle of contamination prevention is to add an appropriate amount of dUTP in the PCR reaction, replacing dTTP with dUTP in DNA, forming PCR amplification products containing the dU base. In subsequent PCR reactions, the UDG enzyme selectively cuts the single or double-stranded DNA containing dU that might have been contaminated from previous PCR amplification, preventing potential contamination effects on the current PCR amplification.

SYBR Green One-Step qRT-PCR Kit (UDG) uses extracted RNA as a template and qPCR primers to continuously perform reverse transcription and fluorescence quantitative PCR in the same reaction tube. This ensures quick, simple operations, minimizing human errors, effectively reducing contamination risk, saving operational time in PCR experiments, and enabling high throughput.

This product integrates efficient M-MuLV Reverse Transcriptase, RNase Inhibitor, and the superior antibody-bound hotstart Taq DNA Polymerase, with an optimized buffer system. It offers excellent reverse transcription performance, high sensitivity, strong amplification specificity, and good reaction stability. It is particularly suitable for the detection of low-abundance endogenous RNA, exogenous viral RNA, and other trace RNAs.

SYBR Green One-Step qRT-PCR Kit (20X, UDG) uses SYBR Green I as a dye. SYBR Green I is a green fluorescent dye that binds to the minor groove region of double-stranded DNA (dsDNA). Its fluorescence is relatively weak when unbound, but greatly intensifies upon binding to dsDNA. Thus, by monitoring fluorescence intensity, one can quantify the amount of dsDNA produced during PCR amplification.

The hotstart Taq DNA Polymerase used in this product is a high-quality, antibody-bound hot start enzyme. The Taq enzyme in hotstart Taq DNA Polymerase binds with a monoclonal antibody against the Taq enzyme, inhibiting the Taq enzyme's DNA polymerase activity. This effectively prevents non-specific annealing of primers and template DNA or primer dimer formation at low temperatures. During the denaturation step of the PCR reaction, the antibody is inactivated by heat, ensuring the Taq enzyme's activity is released only after denaturation. There is no DNA polymerization before denaturation, significantly improving PCR specificity, sensitivity, and quantitative detection accuracy.

This product includes M-MuLV Reverse Transcriptase, hotstart Taq DNA Polymerase, UDG enzyme, PCR Buffer, dNTPs, dUTP, SYBR Green I fluorescent dye, stabilizers, nuclease-free water, ROX (selected based on different fluorescent quantitative PCR machines), and magnesium ions, among other standard components. This makes operation simpler and use more convenient. Users only need to prepare their primers and sample RNA.

ROX Dye

The product offers both Low ROX and High ROX, widely compatible with fluorescent quantitative PCR machines that either do not require ROX or require either Low ROX or High ROX as a correction dye. ROX's role is to correct fluorescence fluctuations unrelated to PCR, minimizing inter-well differences. Such variations might be caused by pipetting errors or sample evaporation. Different fluorescent quantitative PCR machines have varying requirements for ROX; hence users should select high concentration ROX (High ROX), low concentration ROX (Low ROX), or no ROX based on the actual instrument being used.

Storage

Store at -20ºC in a dark place, valid for two years. Avoid repeated freeze-thaw cycles.

Precautions

• Be careful to avoid RNase contamination. Use RNase-free pipette tips and centrifuge tubes.
• The SYBR Green One-Step Enzyme Mix (20X, UDG) contains high-concentration glycerol, which has a high viscosity. Before use, centrifuge briefly to collect the solution at the bottom of the tube, and gently mix with a pipette. Try to avoid creating bubbles during mixing, and then pipette slowly and accurately.
• Ensure that the SYBR Green One-Step Reaction Buffer (2X) is completely thawed and mixed gently by inverting before use.
• If the amplification fragment is long or the RNA structure is complex, pre-treat the template RNA at 65ºC for 5-10 minutes to improve the reverse transcription efficiency.
• This reaction uses the qPCR Reverse Primer as the gene-specific primer for reverse transcription. Do not use Random Hexamer Primer or Oligo(dT) Primer, which are commonly used for cDNA first-strand synthesis.
• Pay attention to the primer annealing temperature. If the annealing temperature is <60ºC, it is recommended to use a three-step PCR amplification.
• This product contains SYBR Green I fluorescent dye. Protect it from strong light exposure during storage and when setting up the PCR reaction to prevent fluorescence quenching.
• For amplification fragments longer than 350bp or with a high GC content, it's recommended to increase the extension time to 60 seconds or use a three-step method to improve the amplification efficiency.
• Testing has shown that repeated freeze-thaw cycles (up to 10 times) have no significant effect on the product's performance. However, avoid repeated freeze-thaw cycles as much as possible, as it may reduce product efficiency.
• qPCR detection is highly sensitive. Although this product offers excellent contamination prevention, the PCR reaction setup area should still avoid any potential contamination from amplifiable products. Discard PCR products in sealed containers to prevent contamination of the experimental environment by high-concentration PCR products.
• This product is for scientific research use by professionals only. It should not be used for clinical diagnosis or treatment, in food or drugs, and should not be stored in regular residences.
• For your safety and health, please wear a lab coat and use disposable gloves when handling.

Related:

• Probe One-Step qRT-PCR Kit (20X, UDG)
• Multiplex Probe qPCR Mix (2X, UDG)
• Probe qPCR Mix (2X, UDG)
• SYBR Green One-Step qRT-PCR Kit (20X, UDG)
• SYBR Green qPCR Mix (2X, UDG)

Only for research and not intended for treatment of humans or animals

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