Can TaqMan™ Gene Expression Assays be used in a multiplex reaction?

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Answer

These assays have not been tested to run in a multiplex environment. If you would like to multiplex TaqMan™ Gene Expression Assays, you can select any of our pre-design assays with a VIC dye label so that you can run two seperate assays in the same well. For information about optimizing a multiplex gene expression experiment, please see "Factors Influencing Multiplex Real-Time PCR". You can get the document by using "Factors influencing multiplex' as a keyword to search our web site.

Answer Id: E1836

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What are your recommendations for the best way to dilute and store my TaqMan™ probe?

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Answer

The probe should be diluted in TE buffer [10 mM Tris-HCl, pH 8.0 (at 25°C), 1 mM EDTA]. We recommend storing your probes, both stocks and working solutions, at -20°C in aliquots. This is to cut down on the number of times the probe is subject to freeze-thaws. For detailed information on diluting primers and probes, please refer to the tutorial entitled "Reconstituting and Diluting Primers and TaqMan™ Probes". You can find it on our website by entering this title in the main search field.

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What are the key features of a TaqMan™ MGB probe?

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TaqMan™ MGB probes have a nonfluorescent quencher at the 3´ end and a minor groove binder at the 3´ end that increases the melting temperature (Tm) of probe, allowing the use of shorter probes.  For more information on MGB probes, please consult the following manual: "Primer Express v1.5 and TaqMan™ MGB Probes for Allelic Discrimination: All PCR Instruments: User Bulletin." You can find a copy on our website by entering this title in the main search box.

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When using the TaqMan™ Gene Signature Plates, should I mix the reactions after adding the master mix and template to the wells containing lyophilized primers and probe?

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Answer

No, but it is recommended that the plate be centrifuged to draw down the liquid to the bottom of the wells.

Answer Id: E1370

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Do you offer a human beta actin probe with a reporter dye other than FAM™ dye?

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Answer

We do offer two VIC™ labeled beta actin probes as part of TaqMan® Endogenous Controls:

1) Human beta actin (VIC™/ TAMRA™ Probe, Primer Limited) Cat. No. 4310881E
2) Human beta actin (VIC™/ MGB Probe, Primer Limited) Cat. No. 4326315E

Please consult our Real-Time PCR Applications page for more information.

Answer Id: E1571

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Can I use the TaqMan™ β-actin Detection Reagents (Cat. No. 401846) as an endogenous control for my gene expression studies?

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Answer

The TaqMan™ β-actin Detection Reagents were designed to amplify genomic DNA, and thus are not recommended for use as an endogenous control for gene expression studies. However, we do offer other TaqMan™ assay reagents that were specifically designed for that application. Two examples are: Human ACTB (Beta Actin) Endogenous Control, Cat. No. 4310881E (TAMRA™ probe) or Cat. No. 4326315E (MGB probe).

There are many factors to consider when validating a specific endogenous control. For additional information on designing gene expression assays and controls, please refer to the following documents: "Protocol: TaqMan™ Human Endogenous Control Plate", and User Bulletin #2: "Relative Quantitation of Gene Expression". You can find copies of these documents on our website by entering the titles in the main Search field.

Answer Id: E1780

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