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

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

Cat. No.: MPQU-2500 (for 2500T)

Description

Multiplex Probe qPCR Mix (2X, UDG) is a new high-quality anti-contamination (Carryover prevention) premix for multiplex real-time fluorescent quantitative PCR based on probes such as TaqMan. It is mainly used for ultra-sensitive specific quantitative detection of cDNA and genomic DNA. This product contains an optimized proportion of high-quality UDG enzyme and dUTP, which effectively eliminates the false positives or low CT values caused by product contamination during PCR amplification. Since TaqMan probes are generally used, this method is also commonly referred to as the anti-contamination multiplex TaqMan probe method.

UDG (Uracil-DNA Glycosylase), also known as UNG (Uracil-N-glycosylase), can catalyze the hydrolysis of the N-glycosidic bond between the uracil (dU) base in the DNA strand containing uracil and deoxyribose, thereby releasing free uracil. It is mainly used to eliminate product contamination problems caused by PCR amplification. Its contamination prevention principle is: by adding an appropriate amount of dUTP in the PCR reaction, replacing dTTP with dUTP in DNA, forming PCR amplification products containing dU bases. In subsequent PCR reactions, the UDG enzyme selectively cuts the single or double-stranded DNA containing dU that may have been contaminated from previous PCR amplifications, thus avoiding the potential contamination of previous PCR products on the current PCR amplification.

This product is particularly suitable for the quantitative or qualitative detection of low abundance or high specificity target genes. For example, highly sensitive detection of low abundance mRNA, lncRNA, small RNA, microbial RNA after reverse transcription, and SNP detection of homologous genes or genes with high similarity that are difficult to distinguish using conventional dye methods.

Principle

The probe method qPCR does not use fluorescent dyes such as SYBR Green to stain PCR products. Instead, it employs a DNA probe labeled with a fluorescent group and a quenching group (quencher) targeting the target sequence to be detected by PCR (the probe binding site is usually located between the two primer binding sites). Normally, the quencher on the probe causes fluorescence quenching due to spatial fluorescence resonance energy transfer (FRET). During the PCR reaction amplification of the target sequence, both primers and probes will anneal to the target gene. With the extension of the primer, the 5'→3' exonuclease activity of the Taq enzyme causes the probe bound to the target sequence to be degraded from the 5' end. After the fluorescent group and quenching group of the probe are cut by the Taq enzyme, the action of the quenching group disappears, and the fluorescent group can be excited by the excitation light to produce fluorescence. With each PCR cycle, more fluorescent groups are released, and the fluorescence intensity is directly proportional to the number of newly synthesized target fragments, allowing for quantitative detection. The probe is usually a linear DNA specific to the target sequence, with a fluorescent group like FAM or HEX at the 5' end, and a fluorescence quenching group like BHQ1, TAMRA, or MGB at the 3' end.

Features

• Good specificity, high sensitivity, and broad compatibility: Specificity depends not only on PCR primers but also on the specificity of the probe. Specific binding and degradation of the probe and target gene can produce a fluorescent signal, and the sensitivity and specificity of detection are usually significantly higher than methods using fluorescent dyes like SYBR Green.
• For multiplex detection: In a single reaction well, different genes correspond to different probes, and different probes correspond to different fluorescent labels, enabling multiplex fluorescent quantitative PCR detection. Tests have shown that, with proper optimization of primers and probes, this product can be used for the detection of 2-4 genes simultaneously. The product is compatible with a wide range of product GC content and primer Tm values.
• Hotstart property: The Hotstart Taq DNA Polymerase used in this product is a high-quality heat-activated enzyme bound to an antibody. It achieves convenient and efficient heat activation. The Taq enzyme in Hotstart Taq DNA Polymerase binds to the monoclonal antibody against Taq enzyme, inhibiting the DNA polymerase activity of the Taq enzyme. This effectively prevents non-specific annealing of primers and template DNA or dimerization of primers at low temperatures. The antibody is inactivated by heating during the pre-denaturation step of the PCR reaction, ensuring that the activity of the Taq enzyme is only released after pre-denaturation. There is no DNA polymerization reaction before denaturation, greatly improving the specificity, sensitivity, and accuracy of PCR reactions and quantitative detection.
• Easy to use: This product includes all common components such as Hotstart Taq DNA Polymerase, UDG enzyme, PCR Buffer, dNTPs, dUTP, stabilizers, and magnesium ions, making operation simpler and more convenient. Users only need to prepare primers, probes, sample DNA, and deionized water.

ROX Dye

This product offers both Low ROX and High ROX, making it broadly compatible with fluorescent quantitative PCR machines that either do not require ROX or require Low ROX or High ROX as a reference dye. The role of ROX is to correct fluorescence fluctuations unrelated to PCR, thereby minimizing inter-well variations. Such variations can be caused by various factors, such as pipetting errors and sample evaporation. Different fluorescent quantitative PCR machines have different requirements for ROX, so please choose either high concentration ROX (High ROX), low concentration ROX (Low ROX), or no ROX Probe based on the specific instrument in use when preparing the reaction system.

Storage

Store in the dark at -20ºC, valid for one year; Store in the dark at 4ºC, valid for one month. Avoid repeated freeze-thaw cycles.

Precautions

• The fluorescence labeling of the probe should be determined based on the fluorescence compatibility of the qPCR machine in use. For fluorescent quantitative PCR machines that require ROX as a correction dye, avoid using ROX-labeled probes.
• When used for multiplex detection, it is necessary to optimize primers and probes properly, and use probes labeled with appropriate different fluorescent groups. After confirming the results, proceed with multiplex detection. It is generally recommended not to exceed four-fold detection.
• Before use, ensure that the entire tube of reagent is completely thawed. Invert gently to mix before using. Avoid introducing air bubbles during the mixing process.
• Pay attention to the primer annealing temperature. When the annealing temperature is <60ºC, a three-step PCR amplification is recommended.
• For amplification fragments exceeding 350bp or with a high GC content, it is advised to extend the elongation time to 60 seconds or adopt a three-step method to improve amplification efficiency.
• Through testing, it has been observed that 10 repeated freeze-thaw cycles have no significant impact on the product's effectiveness. However, repeated freeze-thaw cycles should still be avoided, as they might reduce the product's performance.
• qPCR detection is ultra-sensitive. Even though this product has excellent contamination prevention, the PCR reaction setup area should still avoid any potential contamination from amplifiable products. Dispose of PCR products in a sealed manner to prevent contamination of the experimental environment by high-concentration PCR products.
• This product is exclusively for scientific research by professionals and should not be used for clinical diagnosis or treatment, food, or drugs. It should not be stored in ordinary residences.
• For your safety and health, please wear a lab coat and use disposable gloves when handling.

Related:

• Universal Probe One-Step RT-qPCR Kit
• 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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