Can Power SYBR® Green PCR Master Mix be substituted directly for the SYBR® Green PCR Master Mix?
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: 2486
How can RNA standards be generated to perform absolute quantitation for RNA targets?
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: 1380
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?
What are the key differences between a TaqMan® MGB probe and a TaqMan® TAMRA™ dye-labeled probe?
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: 1416
What is the difference in sensitivity between TaqMan® chemistry vs. SYBR® Green reagent chemistry?
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: 1419