I checked the efficiency of my SYBR™ Green primer set, and it is low. What is causing this?

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Answer

Poor efficiency is typically caused by PCR inhibitors, limiting reagents, or suboptimal assay design. Please see these examples and suggested solutions in our Real-Time Troubleshooting Tool for more details at https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/poor-pcr-efficiency.html. You can also review this short video (https://www.youtube.com/watch?v=T8Ovs6OC4KE).

Answer Id: E7503

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

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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

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My amplification curves have a funny shape in my qPCR experiment. What is causing this?

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There are several reasons that amplification could be delayed. Please see the information 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/abnormal-amplification-curves/amplification-occurs-later.html) for more details.

Answer Id: E7505

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What are the different phases of a qPCR reaction?

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Check out this short video (https://www.youtube.com/watch?feature=player_embedded&v=4sXPUbIrh3A) to understand the different phases of the PCR reaction and why they are important.

Answer Id: E7487

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How do I set the threshold for my qPCR experiment?

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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).

Answer Id: E7508

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When using SYBR™ Green chemistry on an Applied Biosystems™ Real-Time PCR instrument, how do I change settings to reflect that there is no TaqMan™ probe being used in the reaction?

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Refer to the product manual for your instrument and software for specifics, but in general you will want to change the Quencher value to None.

Answer Id: E1398

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

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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

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

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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

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What primer concentration should I use for SYBR™ Green reactions?

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Answer

The optimal primer concentration needs to be determined empirically, but a good starting point is 200-300 nM (each primer). If you are using a master mix, check the manual for specific recommendations.

Answer Id: E7491

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

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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

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How do I set the baseline for my qPCR experiment?

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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).

Answer Id: E7509

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

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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

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

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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

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

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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

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How much first-strand cDNA should be used in a PCR reaction?

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Answer

The volume will depend on the starting amount of RNA used for first-strand synthesis, and the abundance of the target gene. We recommend starting with 5 μL of the first-strand reaction in a 50 μL PCR reaction (i.e., 1/10 volume). More than 10% may inhibit downstream reactions. In general, for gene expression you will want to start with 1-100 ng of cDNA per well.

Answer Id: E7483

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