SYBR™ Green PCR Master Mix, 5 x 5 mL - FAQs

Can I use SYBR Green PCR Master Mix (Cat. No. 4309155, 4364344, 4364346, 4312704, 4334973) in Fast mode?

SYBR Green PCR Master Mix is not supported for use with Fast Mode thermal cycling conditions. When using SYBR Green PCR Master Mix on the StepOne, 7500 Fast, or 7900HT Fast instruments, use Standard mode thermal cycling conditions.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

When using SYBR Green dye chemistry on an Applied Biosystems 7900HT Fast, a 7500 or 7500 Fast, or a 7300 Real-Time PCR System, is there an option to set the quencher to reflect the fact that there is no TaqMan probe being used in the reaction?

Yes. On the Detector Manager for the System, set the Quencher to “Non Fluorescent”.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

Is the use of AmpErase UNG necessary for performing reverse transcription reactions?

No. We do not recommend that you use AmpErase UNG when performing reverse transcription. When using a dNTP mix with dUTP in a RT reaction, uracil will get incorporated into the cDNA generated from your RNA template. AmpErase UNG (uracil N-glycosylase), through an enzymatic reaction, will cleave single or double stranded DNA dUTP containing sequences.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

Can Power SYBR Green PCR Master Mix (Cat. No. 4368577, 4367659, 4368706, 4368702, 4368708, 4367660) be substituted directly for the SYBR Green PCR Master Mix (Cat. No. 4309155, 4364344, 4364346, 4312704, 4334973)?

The Power SYBR Green PCR Master Mix is a new and improved formulation of the regular SYBR Green PCR Master Mix. You may need to verify your assays in order to start using the Power SYBR Green PCR Master Mix. For more information on verifying your reaction with the Power SYBR Green PCR Master Mix, please refer to the Power SYBR Green PCR Master Mix protocol.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

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”.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

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.

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?

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.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

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.