qPCRBIO Genotyping Mix

qPCRBIO Genotyping Mix is designed for fast, accurate and reproducible allelic discrimination for use in TaqMan® and other dual-labelled probe-based genotyping assays.

The optimised buffer system and antibody-mediated hot start technology result in superior allele clustering and accurate characterisation of class I to class IV mutations.

Features

  • Accurate genotype calling
  • Superior allele clustering
  • Antibody-mediated hot start for improved sensitivity
  • Compatible with all real-time PCR platforms

 

CAT No.
Product
Size
Price
QTY
PB20.41-01
qPCRBIO Genotyping Mix Lo-ROX
100 x 20μL Reactions
$50.00
PB20.41-05
qPCRBIO Genotyping Mix Lo-ROX
500 x 20μL Reactions
$240.00
PB20.41-20
qPCRBIO Genotyping Mix Lo-ROX
2000 x 20μL Reactions
$810.00
PB20.42-01
qPCRBIO Genotyping Mix Hi-ROX
100 x 20μL Reactions
$50.00
PB20.42-05
qPCRBIO Genotyping Mix Hi-ROX
500 x 20μL Reactions
$240.00
PB20.42-20
qPCRBIO Genotyping Mix Hi-ROX
2000 x 20μL Reactions
$810.00
PB20.43-01
qPCRBIO Genotyping Mix No-ROX
100 x 20μL Reactions
$50.00
PB20.43-05
qPCRBIO Genotyping Mix No-ROX
500 x 20μL Reactions
$240.00
PB20.43-20
qPCRBIO Genotyping Mix No-ROX
2000 x 20μL Reactions
$810.00

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More Information

qPCRBIO Genotyping Mix is a kit designed for use in TaqMan® and other dual-labelled probe-based genotyping assays. This fast, accurate and reliable mix gives highly reproducible allelic discrimination and is able to accurately call class I to class IV mutations. The mix is compatible with LNA and PNA probes, which offer more stringent allele calling.

SNP detection can be particularly challenging when targets have a high GC or AT content. qPCRBIO Genotyping Mix has been optimised to give tight fluorescence clusters and more accurate allele calling compared to other mixes, even for challenging targets. The mix uses antibody-mediated hot start technology to give improved reaction sensitivity and specificity with the benefit of excellent room temperature stability, ideal for high-throughput genotyping studies.

Applications

  • Genotyping single nucleotide polymorphisms (SNPs)
  • TaqMan® allelic discrimination assays
  • TaqMan® predesigned SNP genotping assays
  • High-throughput genotyping studies

Specifications

qPCRBIO Genotyping Mix Lo-ROX

Component

100 Reactions

500 Reactions

2000 Reactions

2x qPCRBIO Genotyping Mix Lo-ROX

1 x 1 mL

5 x 1 mL

20 x 1 mL

qPCRBIO Genotyping Mix Hi-ROX

Component

100 Reactions

500 Reactions

2000 Reactions

2x qPCRBIO Genotyping Mix Hi-ROX

1 x 1 mL

5 x 1 mL

20 x 1 mL

qPCRBIO Genotyping Mix No-ROX

Component

100 Reactions

500 Reactions

2000 Reactions

2x qPCRBIO Genotyping Mix No-ROX

1 x 1 mL

5 x 1 mL

20 x 1 mL

qPCRBIO Genotyping Mix Lo-ROX

Component

2x qPCRBIO Genotyping Mix Lo-ROX



100 Reactions

1 x 1 mL

500 Reactions

5 x 1 mL

2000 Reactions

20 x 1 mL

qPCRBIO Genotyping Mix Hi-ROX

Component

2x qPCRBIO Genotyping Mix Hi-ROX



100 Reactions

1 x 1 mL

500 Reactions

5 x 1 mL

2000 Reactions

20 x 1 mL

qPCRBIO Genotyping Mix No-ROX

Component

2x qPCRBIO Genotyping Mix No-ROX



100 Reactions

1 x 1 mL

500 Reactions

5 x 1 mL

2000 Reactions

20 x 1 mL

Reaction Volume

Storage

20μL

On arrival, products should be stored between -30 and -15°C. If stored correctly the kit will retain full activity for 12 months.

Reaction Volume

20μL

Storage

On arrival, products should be stored between -30 and -15°C. If stored correctly the kit will retain full activity for 12 months.

Instrument Compatibility

This product is compatible with all standard and fast cycling qPCR instruments. Use our qPCR Selection Tool to find out which ROX variant is compatible with your instrument.

FAQs

Can products generated with qPCRBIO Genotyping Mixes be digested, cloned, and sequenced?

Yes, PCR products generated with qPCRBIO Genotyping Mixes have the same characteristics as PCR products generated with wild-type Taq polymerase. They may be sequenced or digested with restriction endonucleases using standard protocols. Products are 3′-d(A)-tailed and may be used for TA cloning or may be blunt-ended or digested with restriction enzymes prior to cloning. For best results, we recommend purifying the PCR products using any standard PCR clean-up kit.

Can ROX have a negative impact on the reaction?

ROX (6-carboxy-X-rhodamine) is used as a passive reference dye in ROX-dependent real-time PCR instruments to normalize for variations of fluorescence levels that can arise mainly due to optical path variations among wells. Normalisation of the fluorescence intensity (Rn) is done in real-time PCR software by dividing the emission intensity of the specific signal by the emission intensity of ROX.

ROX does not take part in the PCR reaction and its fluorescence levels are not proportional to the quantity of DNA in each well, so the addition of this fluorophore to a mix provides a constant fluorescent signal during amplification.

Different types of real-time PCR instruments requiring a passive reference standard have different optimal concentrations of ROX, mainly due to the different optical configurations of each system (i.e. the different type of excitation source and optics used).

The addition of either too little or too much ROX would result in a very noisy signal impacting on the results of the reaction. Therefore, it is extremely important for the user to:

  1. Determine the correct ROX concentration to optimise real-time PCR results, and
  2. Check the ROX settings on the software used to set up the reaction

A useful selection tool for the most commonly used systems can be found here.

What is included in qPCRBIO Genotyping Mixes?

qPCRBIO Genotyping Mixes are ready to use qPCR 2x Mastermixes. You only need to add primers, template DNA and PCR grade water during reaction set up.

What is ROX?

ROX is a passive reference dye which means it does not take part in the PCR reaction. It is used to normalise non-PCR related fluctuations in fluorescence.

What is the concentration of ROX in qPCRBIO Genotyping Mixes?

The qPCRBIO Genotpying Mixes come in 3 different formulations, each with a different concentration of ROX:

  • qPCRBIO Genotpying Mix Lo-ROX (PB20.41) contains 112 nM ROX.
  • qPCRBIO Genotpying Mix Hi-ROX (PB20.42) contains 1.12 µM ROX.
  • qPCRBIO Genotpying Mix No-ROX (PB20.43) does not contain ROX.

You can use our qPCR Selection Tool under the Resources drop-down menu to determine which of our mixes are best suited for your qPCR machine.

What troubleshooting recommendations are there if no signal is detected?

We recommend optimising the reaction by applying the following:

  • Increase the concentration of the probe. Keep in mind that adding too much probe can slow the PCR reaction as the processivity of the polymerase goes down when the it cleaves the probe. However, doubling the probe concentration should not cause any significant effects. Applying this should increase the fluorescence signal enough to be detected and be higher than the ROX level, if ROX is being used. We advise a titration of probe (10% more, 20% more, etc.) to find a concentration where the probe signal is high enough to be detected or is above the ROX signal, without inhibiting the PCR reaction.
  • Different annealing temperatures (55-67°C) could also be tested. As this is a probe-based assay, an optimisation round will be required for each probe/primer set. To help with testing, 1x SYBRTM dye could be added to the mix so that a melt analysis can be carried out after the PCR. This helps determine if a product was obtained. If no product is obtained, this could mean the probe didn’t anneal properly therefore optimisation of the probe may be required.

Why is the reaction so slow?

The qPCRBIO Genotyping Mix is one of our slowest products. The cycling conditions have been designed this way as this increases the chances of the correct primer binding to the correct allele. If faster conditions are required, consider that the conditions vary significantly based on the probes and how significant the differences are between the alleles. An optimisation trial can be carried out for each desired product to determine the shorter anneal/extension time. If switching from competitors’ products, start the optimisation with the same conditions used for their assay.

More Information

qPCRBIO Genotyping Mix is a kit designed for use in TaqMan® and other dual-labelled probe-based genotyping assays. This fast, accurate and reliable mix gives highly reproducible allelic discrimination and is able to accurately call class I to class IV mutations. The mix is compatible with LNA and PNA probes, which offer more stringent allele calling.

SNP detection can be particularly challenging when targets have a high GC or AT content. qPCRBIO Genotyping Mix has been optimised to give tight fluorescence clusters and more accurate allele calling compared to other mixes, even for challenging targets. The mix uses antibody-mediated hot start technology to give improved reaction sensitivity and specificity with the benefit of excellent room temperature stability, ideal for high-throughput genotyping studies.

Applications

  • Genotyping single nucleotide polymorphisms (SNPs)
  • TaqMan® allelic discrimination assays
  • TaqMan® predesigned SNP genotping assays
  • High-throughput genotyping studies

Specifications

qPCRBIO Genotyping Mix Lo-ROX

Component

100 Reactions

500 Reactions

2000 Reactions

2x qPCRBIO Genotyping Mix Lo-ROX

1 x 1 mL

5 x 1 mL

20 x 1 mL

qPCRBIO Genotyping Mix Hi-ROX

Component

100 Reactions

500 Reactions

2000 Reactions

2x qPCRBIO Genotyping Mix Hi-ROX

1 x 1 mL

5 x 1 mL

20 x 1 mL

qPCRBIO Genotyping Mix No-ROX

Component

100 Reactions

500 Reactions

2000 Reactions

2x qPCRBIO Genotyping Mix No-ROX

1 x 1 mL

5 x 1 mL

20 x 1 mL

qPCRBIO Genotyping Mix Lo-ROX

Component

2x qPCRBIO Genotyping Mix Lo-ROX



100 Reactions

1 x 1 mL

500 Reactions

5 x 1 mL

2000 Reactions

20 x 1 mL

qPCRBIO Genotyping Mix Hi-ROX

Component

2x qPCRBIO Genotyping Mix Hi-ROX



100 Reactions

1 x 1 mL

500 Reactions

5 x 1 mL

2000 Reactions

20 x 1 mL

qPCRBIO Genotyping Mix No-ROX

Component

2x qPCRBIO Genotyping Mix No-ROX



100 Reactions

1 x 1 mL

500 Reactions

5 x 1 mL

2000 Reactions

20 x 1 mL

Reaction Volume

Storage

20μL

On arrival, products should be stored between -30 and -15°C. If stored correctly the kit will retain full activity for 12 months.

Reaction Volume

20μL

Storage

On arrival, products should be stored between -30 and -15°C. If stored correctly the kit will retain full activity for 12 months.

Instrument Compatibility

This product is compatible with all standard and fast cycling qPCR instruments. Use our qPCR Selection Tool to find out which ROX variant is compatible with your instrument.

FAQs

Can products generated with qPCRBIO Genotyping Mixes be digested, cloned, and sequenced?

Yes, PCR products generated with qPCRBIO Genotyping Mixes have the same characteristics as PCR products generated with wild-type Taq polymerase. They may be sequenced or digested with restriction endonucleases using standard protocols. Products are 3′-d(A)-tailed and may be used for TA cloning or may be blunt-ended or digested with restriction enzymes prior to cloning. For best results, we recommend purifying the PCR products using any standard PCR clean-up kit.

Can ROX have a negative impact on the reaction?

ROX (6-carboxy-X-rhodamine) is used as a passive reference dye in ROX-dependent real-time PCR instruments to normalize for variations of fluorescence levels that can arise mainly due to optical path variations among wells. Normalisation of the fluorescence intensity (Rn) is done in real-time PCR software by dividing the emission intensity of the specific signal by the emission intensity of ROX.

ROX does not take part in the PCR reaction and its fluorescence levels are not proportional to the quantity of DNA in each well, so the addition of this fluorophore to a mix provides a constant fluorescent signal during amplification.

Different types of real-time PCR instruments requiring a passive reference standard have different optimal concentrations of ROX, mainly due to the different optical configurations of each system (i.e. the different type of excitation source and optics used).

The addition of either too little or too much ROX would result in a very noisy signal impacting on the results of the reaction. Therefore, it is extremely important for the user to:

  1. Determine the correct ROX concentration to optimise real-time PCR results, and
  2. Check the ROX settings on the software used to set up the reaction

A useful selection tool for the most commonly used systems can be found here.

What is included in qPCRBIO Genotyping Mixes?

qPCRBIO Genotyping Mixes are ready to use qPCR 2x Mastermixes. You only need to add primers, template DNA and PCR grade water during reaction set up.

What is ROX?

ROX is a passive reference dye which means it does not take part in the PCR reaction. It is used to normalise non-PCR related fluctuations in fluorescence.

What is the concentration of ROX in qPCRBIO Genotyping Mixes?

The qPCRBIO Genotpying Mixes come in 3 different formulations, each with a different concentration of ROX:

  • qPCRBIO Genotpying Mix Lo-ROX (PB20.41) contains 112 nM ROX.
  • qPCRBIO Genotpying Mix Hi-ROX (PB20.42) contains 1.12 µM ROX.
  • qPCRBIO Genotpying Mix No-ROX (PB20.43) does not contain ROX.

You can use our qPCR Selection Tool under the Resources drop-down menu to determine which of our mixes are best suited for your qPCR machine.

What troubleshooting recommendations are there if no signal is detected?

We recommend optimising the reaction by applying the following:

  • Increase the concentration of the probe. Keep in mind that adding too much probe can slow the PCR reaction as the processivity of the polymerase goes down when the it cleaves the probe. However, doubling the probe concentration should not cause any significant effects. Applying this should increase the fluorescence signal enough to be detected and be higher than the ROX level, if ROX is being used. We advise a titration of probe (10% more, 20% more, etc.) to find a concentration where the probe signal is high enough to be detected or is above the ROX signal, without inhibiting the PCR reaction.
  • Different annealing temperatures (55-67°C) could also be tested. As this is a probe-based assay, an optimisation round will be required for each probe/primer set. To help with testing, 1x SYBRTM dye could be added to the mix so that a melt analysis can be carried out after the PCR. This helps determine if a product was obtained. If no product is obtained, this could mean the probe didn’t anneal properly therefore optimisation of the probe may be required.

Why is the reaction so slow?

The qPCRBIO Genotyping Mix is one of our slowest products. The cycling conditions have been designed this way as this increases the chances of the correct primer binding to the correct allele. If faster conditions are required, consider that the conditions vary significantly based on the probes and how significant the differences are between the alleles. An optimisation trial can be carried out for each desired product to determine the shorter anneal/extension time. If switching from competitors’ products, start the optimisation with the same conditions used for their assay.