TaqMan™ TAMRA Probe, 6,000 pmol - FAQs

I would like to calibrate my Real-Time PCR instrument with a custom dye, which buffer should I use?

Please ask your dye manufacturer for recommendations. If there is no specific recommendation available, try TE Buffer (pH 8.0) to start.

I need to see all the primer and probe sequences from an assay design right away. How can I do this?

You can directly see all the primer and probe sequences from a test design right away using the Primer Express Software. Open the software and choose File => New. Choose either TaqMan MGB Quantification or TaqMan Quantification for Gene Expression Assay design. Copy/paste your gene sequence of interest into the new window and click on the green triangle to get primer/probe designs.

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

How can I determine whether the Tm of my custom probe is appropriate?

In general, the target Tm of a TaqMan probe is 70 degrees C. Using the Primer Express Software, you can check the Tm of a MGB or TAMRA probe probe.

Within the software, go to Primer Probe Test Tool and then copy/paste in your sequence of interest to see the Tm. If you need to check for a MGB based probe, make sure to choose Document Type: TaqMan MGB Quantification. If you need to check for a QSY or TAMRA based probe, make sure to choose Document Type: TaqMan Quantification. Real-Time PCR and Digital PCR Applications

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

I have purchased custom oligos (primers and probes) and I want to make a 20x working stock for a TaqMan assay, similar to the pre-designed assays. How do I dilute and mix my oligos to make a 20x working stock?

Most pre-designed TaqMan assays are supplied as a 20x assay concentrate containing 18 uM of each primer and 5 uM of probe which provides a 900 nM of each primer and 250 nM of the probe (for gene expression assays) and 200 nM of each probe (for SNP genotyping assays) at the final 1x concentration.

In order to make a 20x working stock from custom oligos, the oligos need to be diluted as follows: To make a 20x Assay stock for Gene Expression assays, make 100 uM stocks of both forward and reverse primers, and TaqMan probe. Add 18 ul of each primer and 5 ul of the TaqMan probe, which gives 41 ul volume. Add 59 ul of 1X TE to bring the volume to 100 ul of 20X TaqMan Gene Expression Assay. To make a 20x Assay stock for a Genotyping Assay, make 100 uM stocks of both forward and reverse primers, and both TaqMan probes. Add 18 ul of each primer and 4 ul of each TaqMan probe, which gives 44 ul volume. Add 56 ul of 1X TE to bring the volume to 100 ul of 20X TaqMan SNP Genotyping Assays.

I am looking to order a molecular probe for validation, using specific sequences. How do I order a custom probe?

We do not offer any custom probes for diagnostic purposes.

We do offer custom TaqMan probes for qPCR experiments, which are for Research Use Only.
TaqMan MGB Probe
TaqMan QSY Probe
TaqMan TAMRA Probe
These above probes can be designed in our online tool, Custom TaqMan Probes.

If you would like to order a pre-mix of primers and probe, please check our custom TaqMan Assays.
You can find more information one the Custom TaqMan Gene Expression Assay page on thermofisher.com.

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

I received a shipment of TaqMan probes but I forgot to store them at -20 degrees C and they stayed at room temperature over the weekend. Can you please confirm if the probes are still functional?

TaqMan probes are recommended to be stored at -20 degrees C, but they can be subjected to higher temperature for several days and still retain their quality and stability. Please have a look at our White Paper: Custom Primers and TaqMan Probes shipped at ambient temperature reduce environmental impact and retain their quality and stability, for more details: https://assets.thermofisher.com/TFS-Assets/LSG/brochures/PrimersProbes-ambient-shipping-WhitePaper.pdf

Find additional tips, troubleshooting help, and resources within our TaqMan Primers and Probes Support Center.

How do I analyze a real time run on an ABI PRISM 7700 Sequence Detection System in which the run contains reactions that have both TaqMan MGB probes and TaqMan TAMRA dye labeled probes?

If the TaqMan MGB probes are in separate tubes from the TaqMan TAMRA dye-labeled probes, you should analyze the data sets separately. For the TaqMan TAMRA dye-labeled probe, analyze as usual. For the TaqMan MGB probe you need to uncheck the Quencher box in the Sample Type Setup, and then analyze. This would be done after the run is completed. If you are multiplexing with a TaqMan MGB and a TaqMan TAMRA dye-labeled probe in the same reaction, it is recommended to leave the quencher as TAMRA dye for analysis.

What are the differences in the various types of purification possible when ordering custom oligos?

OPC: OPC (Oligonucleotide Purification Cartridge) purification is based on the principle of reverse phase chromatography. Oligonucleotides which are to be purified by OPC are synthesized trityl-ON. The trityl (DMT) group at the 5' terminus is hydrophobic, which allows selective retention when the oligonucleotide is added to the cartridge. Truncated failure sequences do not contain a 5' DMT group and are therefore flushed through the cartridge. The retained oligonucleotide is then detritylated and recovered in aqueous organic solvent (20% acetonitrile). This is used to desalt the oligo. It's a way to precipitate the DNA from solution.

HPLC purification: Reverse phase HPLC is used for the purification of oligos. This is the type of purification that will give you the most purity. This type of purification can be requested with large scale oligos.

Can I replace a TaqMan probe with fluorescent primers?

No, fluorescent primers will not function in the same manner as a TaqMan probe. We do not support any applications using fluorescent primers on our Real-Time PCR platforms.

How can I calculate the concentration of a TaqMan probe?

The initial concentration or total yield of your synthesized probe is listed on the data sheet supplied with the probe. TaqMan probes are sometimes shipped in the lyophilized state, but more often are shipped in solution (1X TE). To create a working stock solution of your probe or primers, you should reconstitute or dilute the probe in the appropriate volume of sterile 1X TE (1 mM Tris, 0.1 mM EDTA, pH 8.0) or sterile, nuclease-free H2O. Detailed instructions on how to calculate the proper volume of solution to add to your probe are provided in our online Tutorial document "Reconstituting and Diluting Primers and TaqMan Probes". You can find a copy on our website by entering this title in the main Search field.

Why does a TaqMan TAMRA probe need to have more C's (Cytosines) than G's (Guanidines) in the sequence?

Empirical data has shown that TaqMan TAMRA probes which contain more Gs than Cs will generally have lower ΔRn values and overall poor performance compared to those that do not. For more information on designing TaqMan TAMRA probes, please consult tutorials related to the Primer Express software.

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

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.

Find additional tips, troubleshooting help, and resources within ourTaqMan Primers and Probes Support Center.

What is the advantage of using VIC dye as a reporter instead of JOE dye for a TaqMan probe?

The main benefits are the following:

1) VIC dye is two to three times brighter than JOE dye. Therefore, VIC dye provides for better sensitivity in a TaqMan reaction.

2) VIC dye can be synthesized in all three synthesis scales.

3) The spectral emission of probes labeled with VIC dye is narrower than that of JOE dye, therefore, there is greater distinction between labeled probes in applications that require multiplexing.