You searched for: 

4367659

Product FAQ

Can I do a melt curve with a TaqMan™ assay?

Answer

No. A TaqMan™ probe, once cleaved, cannot be re-quenched. Therefore a melt curve does not apply when using a TaqMan™ assay.

Answer Id: E7493

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Can Power SYBR™ Green PCR Master Mix be substituted directly for the SYBR™ Green PCR Master Mix?

Answer

The Power SYBR Green PCR Master Mix (P/N 4368577/ 4367659/ 4368706/ 4368702/ 4368708/ 4367660) is a new and improved formulation of the regular SYBR Green PCR Master Mix (P/N 4344463/ 4309155/ 4364344/ 4364346/ 4312704/ 4334973). This new master mix contains a more purified AmpliTaq™ enzyme and improved buffer formulation compared to regular SYBR™ Green PCR Master Mix. You may need to re-validate your assays in order to start using the Power SYBR™ Green PCR Master Mix. For more information on validating your reaction with the Power SYBR™ Green PCR Master Mix, please refer to the Power SYBR™ Green PCR Master Mix Protocol (P/N 4367218).

Answer Id: E2486

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What is the difference between absolute quantification (AQ) and relative quantification (RQ)? How do I choose which method to use?

Answer

Absolute quantification will quantitate unknowns based on a known quantity. It involves the creation of a standard curve from a target of known quantity (i.e., copy number). Unknowns can then be compared to the standard curve and a value can be extrapolated. Absolute quantification is useful for quantitating copy number of a certain target in DNA or RNA samples. The result usually is a number followed by a unit, such as copy number and ng, etc.

Relative quantification can quantitate a fold difference between samples. It involves the comparison of one sample to another sample (calibrator) of significance. For example, in a drug treatment study you could compare a treated to an untreated sample. The quantity of the calibrator is not known and cannot be measured absolutely. Therefore the calibrator (untreated sample) and samples (treated samples) are normalized to an endogenous control (a gene that is consistently expressed among the samples) and then compared to each other to get a fold difference. Relative quantification is useful for quantitating messenger RNA levels. Since the result is a fold change or ratio, it is not followed by a unit.

The method that you choose will depend on the type of data you need from your experiment. You can find more information here (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/absolute-vs-relative-quantification-for-qpcr.html) as well.

Answer Id: E7494

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What are the requirements for a standard curve qPCR experiment?

Answer

In a standard curve experiment, you must generate a standard curve for each target gene. The standards should closely represent the sample (i.e., RNA for RNA input, plasmid or gDNA for DNA input). This reference (http://www.ncbi.nlm.nih.gov/pubmed/11013345) is a good review of standard curves and the experimental setup. You can also review this short video (https://www.youtube.com/watch?v=mE5ieko9_RQ) on standard curve experiments.

Answer Id: E7495

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

How many replicates do I need to run for my qPCR experiment? What recommendations do you have for plate setup?

Answer

Please view this short video (https://www.youtube.com/watch?v=eIaPGhOjBQo), which explains some best practices for replicates and plate setup.

Answer Id: E7489

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What are the requirements for a relative quantification qPCR experiment?

Answer

In a relative quantification experiment, you will need to identify an endogenous control and a reference (or calibrator) sample. An endogenous control is a gene that does not change in expression across all the samples in your study. A reference sample is the sample that you are comparing all others to. This is often the untreated, or control, sample. Please see our Relative Gene Expression Workflow bulletin (https://tools.thermofisher.com/content/sfs/brochures/cms_075428.pdf) for more step-by-step guidelines on how to design your experiment.

Answer Id: E7496

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

I’m trying to decide between purchasing a one-step or two-step RT-PCR kit. Can you review the advantages and disadvantages of each?

Answer

One-step RT-PCR is convenient, and less prone to contamination as there is less opportunity for pipetting error. This method is also faster than two-step. However, the cDNA cannot be archived, and fewer genes can be analyzed. Two-step RT-PCR gives you the ability to archive cDNA, analyze multiple genes, and gives greater flexibility. This table (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/1-step-vs-2-step.html) also provides a comparison.

Answer Id: E7486

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What is the difference in sensitivity between TaqMan™ chemistry vs. SYBR™ Green reagent chemistry?

Answer

Sensitivity can actually be equivalent when using TaqMan™ chemistry and SYBR™ Green reagent chemistry. It might seem that a TaqMan™ assay with fluorescent signal generated by a sequence-specific probe would always be more sensitive than a SYBR™ Green reagent assay, but a poorly designed TaqMan™ assay could theoretically be less specific than a well-designed SYBR™ Green reagent assay. However, the potential for detection of primer dimers and non-specific products using SYBR™ Green chemistry is more likely to result in loss of sensitivity when attempting to quantitate lower copy numbers.

For more information on Real-Time PCR chemistries, please refer to the following Application Notes, which you can find on our website through Technical Resources, or by entering the titles in the main Search field: "Real-Time PCR Vs. Traditional PCR", "Essentials of Real Time PCR", and "Selection of Reagents for Real-Time PCR".

Answer Id: E1419

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

How do I measure how much cDNA I have after the reverse transcription?

Answer

It is not possible to directly measure the cDNA concentration by UV absorbance because the dNTPs from the reverse transcription step will also absorb and throw off the measurement. For an accurate reading, you would have to purify the cDNA first. Since this would lead to a loss of material, cDNA concentrations are estimated based on the initial RNA input instead. For example, if you used 100 ng of total RNA in a 20 μL reverse transcription reaction, you can assume a maximum of 100 ng of cDNA (at 5 ng/μL).

Answer Id: E7500

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What should I do if I have extra peaks in my melt curve? What does this mean?

Answer

Since SYBR™ Green can bind to any double-stranded DNA product, it is important to check your melt curves for the number of peaks. When primers are specific, you should see only one peak. Sometimes you may get extra peaks in a melt curve, which could be due to primer-dimers, a nonspecific product, or gDNA contamination. Please watch this short video (https://www.youtube.com/watch?feature=player_embedded&v=4QPyVcpbvNw) for more information on multiple peaks in melt curves and how to deal with them.

Answer Id: E7502

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What can I do if the amplification of my target gene is later than expected for my qPCR experiment?

Answer

There are several reasons that amplification could be delayed. Please see the information in our Real-Time Troubleshooting Tool for more details (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/abnormal-amplification-curves/amplification-occurs-later.html).

Answer Id: E7504

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What is the difference between TaqMan™ and SYBR™ Green methods of detection?

Answer

TaqMan™ and SYBR™ Green chemistries are two different methods of detection for qPCR. Please see this detailed comparison of these two approaches (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/taqman-assays-vs-sybr-green-dye-for-qpcr.html). You can also watch this short video (https://www.youtube.com/watch?feature=player_embedded&v=fkUDu042xic) on how TaqMan™ assays work.

Answer Id: E7490

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

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

Answer

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)

Answer Id: E7510

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

I am getting amplification in my no-template control (NTC) wells in my qPCR experiment. What is causing this?

Answer

There could be several reasons for amplification in a NTC well. 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/amplification-no-template-control.html) for more details.

Answer Id: E7506

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Do I have to normalize my samples for comparative Ct experiments?

Answer

Comparative Ct experiments use an endogenous control gene to normalize the cDNA input. Please watch this short video (https://www.youtube.com/watch?feature=player_embedded&v=jst-3hD_xFQ) for more details on how this works. For a protocol workflow, please refer to our Guide to Performing Relative Quantitation of Gene Expression (https://tools.thermofisher.com/content/sfs/manuals/cms_042380.pdf).

Answer Id: E7497

Was this answer helpful?

Yes
No
Thank you for your response