Detection Enhancer, for real-time PCR
Detection Enhancer, for real-time PCR
Applied Biosystems™

Detection Enhancer, for real-time PCR

Detection Enhancer is uniquely formulated to improve template amplification in real-time PCR (qPCR) workflows. This reagent is particularly beneficial withMás información
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Número de catálogoN.º de reacciones
A448111000 x 25 μL Reactions
A44941500 x 25 μL Reactions
A44810100 x 25 μL Reactions
Número de catálogo A44811
Precio (MXN)
-
N.º de reacciones:
1000 x 25 μL Reactions
Detection Enhancer is uniquely formulated to improve template amplification in real-time PCR (qPCR) workflows. This reagent is particularly beneficial with DNA samples containing GC-rich sequences and/or persistent secondary structure. Detection Enhancer is a component of the AgPath-ID One-Step RT-qPCR Reagents kit and is offered here as a stand-alone product that can be used with any Applied Biosystem real-time PCR master mix.

• Improve qPCR amplification of templates containing high GC content and persistent secondary structure
• Compatible with any Applied Biosystem real-time PCR master mix
• Flexible kit sizes for a range of laboratory throughputs

GC-rich samples
GC-rich DNA sequences are usually defined as those that contain >60% cytosine (C) or guanine (G). GC-rich samples can be particularly challenging to amplify because the sequences are more stable than those with lower GC content. In qPCR experiments, this results in a higher DNA melting point. In addition, GC-rich sequences can form secondary structure, which can add further amplification challenges. Adding a small volume of Detection Enhancer to your reaction mix can dramatically improve qPCR amplification in GC-rich samples.

Enhancing amplification
Detection Enhancer is specifically formulated to address qPCR amplification in particularly stable DNA sequences. It is only for use in real-time PCR workflows where amplification is affected by templates that are GC rich and/or contain persistent secondary structure. The reagents may affect sensitivity for other target types, so it is recommended to first assemble the reaction without Detection Enhancer. If no signal is observed from samples that are expected to be positive, then add Detection Enhancer to the reaction.

The recommended volume of Detection Enhancer per 25 μL reaction is 1.67 μL. For more information on applications of Detection Enhancer, please see the AgPath-ID One-Step RT-qPCR Reagents kit user guide.
Para uso exclusivo en investigación. No apto para uso en procedimientos diagnósticos.
Especificaciones
Para utilizar con (equipo)Sistema 7500
N.º de reacciones1000 x 25 μL Reactions
Línea de productosApplied Biosystems
Tipo de productoDetection Enhancer
Cantidad1000 reactions
Tipo de muestraViral Samples
Condiciones de envíoDry Ice
Volumen2.4 mL
Método de detecciónSonda de cebado
Para utilizar con (aplicación)Real Time PCR (qPCR)
GC-Rich PCR PerformanceHigh
Método de PCR1-step RT-qPCR
Velocidad de reacciónEstándar
Unit SizeEach
Contenido y almacenamiento
2 x 1.2 mL Detection Enhancer, store at -5 to -30°C

Preguntas frecuentes

With the AgPath-ID One-Step RT-PCR Reagents, I am getting signal detection in the no-template control (NTC) reaction. Why is this?

This is likely due to PCR contamination. Here are some recommendations:

- Repeat the qRT-PCR reaction with fresh reagents and decontaminated pipettors.
- Set up and run the qRT-PCR reaction in an area that is isolated from areas used for nucleic acid isolation and PCR product analysis.
- The Reverse Transcriptase enzyme contained in this kit is produced using an E. coli expression vector containing a proprietary version of the MMLV pol gene (GenBank accession no. J02255) expressed from pET-24(+). It is possible that a minimal amount of the expression vector could be carried over into the final mastermix formulation. If you are targeting MMLV, a related virus, or any of the plasmid sequence, we recommend designing primer sequences for target sequences not contained in the expression vector.

What can I do to improve the sensitivity of my qPCR assay?

If you are targeting a low-abundance gene, you may have trouble getting Ct values in a good, reliable range (Ct > 32). To increase the sensitivity of the assay, you may want to consider the following:

- Increase the amount of RNA input into your reverse transcription reaction, if possible
- Increase the amount of cDNA in your qPCR reaction (20% by volume max)
- Try a different reverse transcription kit, such as our SuperScript VILO Master Mix, for the highest cDNA yield possible
- Consider trying a one-step or Cells-to-CT type workflow (depending on your sample type)

How do I set the baseline for my qPCR experiment?

Most times your instrument software can automatically set a proper baseline for your data. Check out our short video, Understanding Baselines, for more information on how to set them (https://www.youtube.com/watch?feature=player_embedded&v=5BjFAJHW-bE).

How do I set the threshold for my qPCR experiment?

In most cases your instrument software can automatically set a proper threshold for your data. Check out our short video, Understanding Thresholds, for more information on how to set them (https://www.youtube.com/watch?feature=player_embedded&v=H_xsuRQIM9M).

I am not getting any amplification with my TaqMan Assay or SYBR Green primer set. What is causing this?

There could be several reasons for no amplification from an assay or primer set. Please see these examples and suggested solutions in our Real-Time Troubleshooting Tool (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/no-amplification.html) for more details.