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

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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

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9edaead1dc37af3e31b6d4dde1809acf_FAQ

How can RNA standards be generated to perform absolute quantitation for RNA targets?

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It is generally not possible to use DNA as a standard for absolute quantitation of RNA because there is no control for the efficiency of the reverse transcription step. Therefore, in-vitro transcribed RNA is commonly used to prepare standards for the absolute quantitation of RNA targets. This would involve the cloning of the target of interest into an in-vitro transcription plasmid, performing in-vitro transcription, then purifying the resulting cRNA so that the DNA plasmid cannot serve as a PCR template. Concentration is measured by A260 and converted to the number of copies using the molecular weight of the RNA.

Relative quantitation of gene expression methods require less up-front preparation and provide a fold-change value instead of an absolute quantity result. For many researchers, absolute quantities are not a necessary parameter to measure, and therefore relative quantitation is a much more attractive approach to studying gene expression via real-time PCR. For more information on relative quantitation of gene expression, please refer to our Technical Reference Library in the Technical Resources section of our website.

Answer Id: E1380

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c701566c3141deca06ee85fb9a0fbf0d_FAQ

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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0f0bf5da8db251bc9b3ce36462b52776_FAQ

What are the key differences between a TaqMan® MGB probe and a TaqMan® TAMRA™ dye-labeled probe?

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Answer

The TaqMan® MGB probes contain the following features:
1) A fluorescent reporter at the 5' end
2) A nonfluorescent quencher at the 3' end. Because the quencher does not fluoresce, the real-time instruments can measure the reporter dye contributions more precisely.
3) A minor groove binder at the 3' end. The minor groove binder increases the melting temperature (Tm) of probes, allowing the use of shorter probes.

In general, the TaqMan® MGB probes exhibit great differences in Tm values between matched and mismatched probes, which provides more accurate allelic discrimination and makes for a more sensitive real-time assay. Mismatches between a probe and allele, or target, reduce the efficiency of probe hybridization in a measurable way, which is especially important in SNP Genotyping assays. Furthermore, AmpliTaq Gold® DNA polymerase is more likely to displace the mismatched probe rather than cleave it to release reporter dye. More information about TaqMan® MGB probes can be found in the User Bulletin entitled "Primer Express Version 1.5 and TaqMan® MGB Probes for Allelic Discrimination." You can find a copy on our website by entering this title in the main search field.

Answer Id: E1416

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ff4fc94b483381baae9b065a9c4c83df_FAQ

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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1fd04d704a1dac1b8878f7f93c10ebee_FAQ

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

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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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9b2a12a64bbd39062237f75c25eec153_FAQ

Can I use my SYBR® Green primers for a TaqMan® assay?

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Answer

It may be possible to use your SYBR® Green primers for a TaqMan® assay, depending on how they were designed. You would have to design a separate probe to use with your existing primers. Please refer to the guidelines in this manual (https://tools.lifetechnologies.com/content/sfs/manuals/cms_041902.pdf) on “Manually Designing Primers and Probes” for the next steps. If you have Primer Express® Software, you can use that software to design a probe. Please note that restricting the design using the predesigned SYBR® primers may not allow for a successful probe design.

Answer Id: E7501

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8d0d3b2fc8aba209b92caec2e403e6bf_FAQ

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

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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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01c20a8eb795cc9c946324a4d3bc487b_FAQ

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.lifetechnologies.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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d346d75fcadba596fde5c2f3e62c1c36_FAQ

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.lifetechnologies.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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c51a54b4df93875c6f9324dbcbd41313_FAQ

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.lifetechnologies.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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b57449628abeab817760685e6bac5ad7_FAQ

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

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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.lifetechnologies.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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9a8ec7489f039a4fa906a66d9bbf87ce_FAQ

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

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Answer

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.lifetechnologies.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.

Answer Id: E7507

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0583f29b66871620fff78188d83ba2a6_FAQ

How do I set the threshold for my qPCR experiment?

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Answer

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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ec5aa87f49608ce3a9ade59c5603b287_FAQ

How do I set the baseline for my qPCR experiment?

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Answer

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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