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.

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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.

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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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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.

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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".

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1fd04d704a1dac1b8878f7f93c10ebee_FAQ

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

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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.

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43aaf1edc23cc652e148336ff9636920_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?

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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.lifetechnologies.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/1-step-vs-2-step.html) also provides a comparison.

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6430b7e052bd115213a6ffb88b138a7c_FAQ

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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a99b7c4d74c4231a90c9c4c20c8d11f6_FAQ

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

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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

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14b0e3b5deae40532f34cc76e015b26e_FAQ

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

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TaqMan® and SYBR® Green chemistries are two different methods of detection for qPCR. Please see this detailed comparison of these two approaches (https://www.lifetechnologies.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.

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700db17c12338327cb7d898d0fe005ca_FAQ

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

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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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1655ec279056d77faf86b2417613a8c7_FAQ

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

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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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9da1f7ccdca89a729c47cfd780d3d6a7_FAQ

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

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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.lifetechnologies.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/absolute-vs-relative-quantification-for-qpcr.html) as well.

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3ce2716407e6cdaf14dfa345480e0c89_FAQ

What are the requirements for a standard curve qPCR experiment?

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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.

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ecab94794ccd473c73565757c1ef9aee_FAQ

What are the requirements for a relative quantification qPCR experiment?

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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.lifetechnologies.com/content/sfs/brochures/cms_075428.pdf) for more step-by-step guidelines on how to design your experiment.

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077028ea7132ed01167101897b7bd651_FAQ