Custom TaqMan™ Gene Expression Assay, FAM, S (360 reactions), made to order - FAQs

I want to order a TaqMan Gene Expression Assay that you have discontinued. Is there a way do that?

Yes, you can order it as a custom assay. Since the assay is discontinued, it can not be ordered on-line. You will have to call or email the order to genomics@appliedbiosystems.com, and include the assay ID. The assay will be synthesized once the order is received. It will be designed and priced as a Custom TaqMan Gene Expression Assay so you will need to choose the scale.

When I click on a TaqMan Gene Expression assay ID, the results page shows that it detects several RefSeq and/or GenBank transcripts. Does this mean the assay detects different genes?

No, the assay is not detecting different genes. Genes often have multiple valid accession numbers, even for the exact same sequence. Even though there are different ID numbers listed, they should all represent the same gene.

What is the date printed on the label of the TaqMan Gene Expression Assays?

The label on the TaqMan Gene Expression Assays lists the expiration date, which under proper storage conditions is 5 yrs. The assay itself is guaranteed for 1 yr upon shipmentt.

When I search for a particular TaqMan Gene Expression Assay, there are multiple assay IDs. What are the differences between them?

We often design multiple assays for the same target. The difference between the assay IDs is most often that the assay is designed across a different exon-exon boundary of a particular transcript sequence. You can confirm the exon-exon boundary by clicking on the individual Assay ID and then look at the Interrogated Sequence Table.

Another difference between assays for the same gene is that they detect different mRNA species or splice variants for the same gene. Ensure that the assay you select will detect your transcripts of interest.

Can I get a custom TaqMan Gene Expression Assay synthesized with different reporter dyes?

No, the Custom TaqMan Gene Expression Assays are only synthesized with FAM dye as the reporter dye at the 5' end and MGB-NFQ as the quencher at the 3' end. Dye selection is only possible with general custom oligo synthesis.

In order to take advantage of our superior in-house assay design algorithm, we recommend that you order a small scale of the standard Custom TaqMan Gene Expression Assay with the FAM label first and validate the assay in a single-plex reaction. You can then submit the sequences of the primers and probes for custom oligo synthesis where you will have the flexibility of choosing your reporter dye.

Does a TaqMan Gene Expression Assay for one species cross-react with another species if there is high degree of sequence homology?

For the 23 species that we cover, you can use the species filter to narrow your results. This is located in the right-hand column of your assay search results. Look for “Exclude Assays that Detect”. Only species that will cross-react are listed (of the 23 species we screen against). Check the species box to remove assays that would detect in that particular species.

I received an email saying that the Custom TaqMan Gene Expression Assay I ordered failed manufacturing. What does this mean and what can be done?

After the primers and probe are designed for a Custom TaqMan Gene Expression Assay, they will be sent for synthesis. Certain primer and probe sequences cannot be synthesized to pass our strict Quality Control procedure. We will try one more time to synthesize the oligos. If the second synthesis fails an email will be sent, notifying you that the order failed manufacturing. You will not be billed if the assay is not manufactured.

If your assay fails during manufacturing you will need to design a new set of primers/probe and send the oligo sequences to us for custom oligo synthesis. If you have an Applied Biosystems real-time PCR instrument, you should have a copy of Primer Express software, which can be used for general primer/probe design.

Can I set up and store the TaqMan Fast Universal PCR Master Mix at room temperature if the instrument is in use?

For optimal results, we recommend to run the reaction plate right after setting up the reaction. If a reaction plate cannot be run within 2 hours of completing the reaction setup, then freeze/refrigerate the reaction plate until it can be loaded and run on the fast PCR instrument platform.

Can I run a reaction made with the standard TaqMan Universal PCR Master Mix in fast thermal cycling mode?

No, we do not recommend this approach. Since the standard TaqMan Universal PCR Master Mix is not optimized for running with fast thermal cycling, assay results will be significantly compromised.

Is the TaqMan Fast Universal PCR Master Mix offered with AmpErase UNG?

The TaqMan Fast Universal PCR Master Mix is provided without any UNG and you should add in AmpEraseUNG to reactions separately, with the addition of an initial 2 minute cycling step at 50º C. If you prefer to have a master mix with the UNG pre-added, please use TaqMan Fast Advanced Master Mix.

Can I compare performance of assays between the TaqMan Fast Universal PCR Master Mix compare and TaqMan Universal PCR Master Mix?

In general, assays have been found to perform comparably with these reagents, but they may not provide exactly identical results.

What are the storage conditions and the shelf life of the TaqMan Fast Universal PCR Master Mix?

We recommend a storage temperature of 4C and the shelf life is 9 months from the manufacturing date.

What is the recommended input amount of template for use with the TaqMan Fast Universal PCR Master Mix?

We recommend adding 10 to 100 ng of RNA converted to cDNA as your template.

Is there a TaqMan PCR Core Reagents kit for the Fast Real-Time PCR System?

No. The only Fast reagents that we offer are the TaqMan Fast Universal PCR Master Mix (2X) for single plex use, and Fast Advanced Master Mix for greater sensitivity and multiplexing use.

Can I use TaqMan Fast Universal PCR Master Mix for both one-step and two-step RT-PCR?

TaqMan Fast Universal PCR Master Mix is recommended for two-step RT-PCR, but for one-step reactions, we recommend using TaqMan Fast Virus 1-Step Master Mix as the preferred reagent.

What component in the TaqMan Fast Universal PCR Master Mix (P/N 4352042) allows rapid amplification results?

The hot-start DNA polymerase enzyme system in the TaqMan Fast Universal PCR Master Mix does not require an activation step, and can be activated with significantly shorter hold times for the thermal cycling stages of PCR, thereby minimizing PCR run time. For more information on using this reagent, please consult the TaqMan Fast Universal PCR Master Mix (2X) protocol.

What is the lowest copy number that can be used with the TaqMan Fast Universal PCR Master Mix (P/N 4352042)?

The fast master mix has demonstrated detection down to 10 copies of the RNase P gene with genomic DNA. Of course, detection limits may vary depending on factors such as assay design and sample preparation.

Which TaqMan assay types can be used when using the with TaqMan Fast Universal PCR Master Mix (P/N 4352042)?

The following research assays are supported using the TaqMan Fast Universal and/or Advanced Fast PCR Master Mix: TaqMan Gene Expression Assays, Custom TaqMan Gene Expression Assays, Custom primers and probes designed from Primer Express software-designed quantitative assays.

How can I obtain the primer and probe sequences for a custom TaqMan Gene Expression Assay that I designed using the Custom Assay Design Tool?

The primer and probe sequences for your custom TaqMan Gene Expression Assay can be found in the Assay Information File (AIF) which can be downloaded here. Once there, select the option "TaqMan™ or Absolute Q™ Assay" and, on the next page, enter the Sales Order number and the Rack/Plate ID for the respective assay (these numbers can be found on the assay packaging near the barcode), and submit. From there you can download a folder including a txt-file with all sequences.
Please note that the sequences are only accessible once the assays have been received.

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

What are the concentrations of the pre-formulated assay mix for the Custom TaqMan Gene Expression Assay, FAM?

Please see the TaqMan Gene Expression Assays - single-tube assays User Guide , page 8.

How do I select a TaqMan Gene Expression Assay for an endogenous control in relative quantitative PCR (qPCR)?

An endogenous control gene is expressed at a consistent level in all studied conditions. It is impossible to predict exactly how any gene will behave under a given range of conditions. The best way of selecting the most appropriate control gene for a relative qPCR experiment is to select some candidate genes and determine their expression levels across the range of experimental conditions and treatments. The genes most stably expressed across these conditions will be the most appropriate controls. See the following links on selection and usage. Check the TaqMan Endogenous Control Assay Selector Tool here.

Find additional tips, troubleshooting help, and resources within our Gene Expression Support Center

Can I perform a one-step RT-PCR reaction using TaqMan Gene Expression Assays?

While TaqMan Gene Expression Assays can be used to perform a one-step RT-PCR reaction, these assays have not been optimized for this application. TaqMan Gene Expression Assays have been designed, tested, and optimized using Applied Biosystems universal conditions for a two-step RT-PCR reaction.

Can gene expression assays designed using the Custom TaqMan Gene Expression Assays Service (Assays-by-Design Service) be multiplexed?

The assays have not been optimized for multiplex, so we don't recommended that you use them in a multiplex reaction. If you would like to try to multiplex, follow the guidelines stated in the Application Note "Factors Influencing Multiplex Real-Time PCR".

I can't match my submitted sequence to any of the probe sequences on the CD Assay Information File (AIF) sent with my Custom TaqMan Assay primers and probe(s). What could be the problem?

Since the Custom TaqMan Assay design software will design oligos to either strand of the target sequence, it is possible that the probe was designed for the complementary strand. Check both strands of sequence for matches to the primer/probe sequences, making sure to check the reverse complement sequence, as well. The assay information file does state "Forward" or "Reverse" to indicate which strand was used to design the probe.

How many replicates are recommended per run for a TaqMan or Custom TaqMan Gene Expression reaction?

We recommend performing four replicates of each sample.

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

I seem to have misplaced the CD for my custom TaqMan assay I recently ordered. How can I get the details?

The details for custom TaqMan assays can be downloaded at: https://www.thermofisher.com/order/taqman-files. Select the option "TaqMan Assays" and, in the screen that follows, indicate the sales order number and the Rack ID of your assay.
Both these numbers can be found on the assay package, next to the barcode.

Why does the amplification curve show weak amplification for the samples in my Custom Gene Expression Assay?

The sequence provided for the assay design may contain mismatches with sample sequences. We recommend that you perform bioinformatics analysis for assay design.
For more information, see the Custom TaqMan Assays Design and Ordering Guide (Pub.No. 4367671).

How do I get the assay information file for my Custom or Custom Plus TaqMan RNA assay?

For orders placed after March 2016, assay information files can be accessed from this page: www.thermofisher.com/taqmanfiles

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

How many reactions can I get out of the Custom and Custom Plus TaqMan Gene Expression assays?

The number of reactions will depend on your final reaction volume. The Custom and Custom Plus TaqMan RNA assays are supplied with the following volumes:
Small: 360 µL (20X)
Medium: 750 µL (20X)
Large: 976 µL (60X)

Assuming a 20 µL final reaction volume, the small size would provide 360 reactions, the medium size would provide 750 reactions, and the large size would provide 2,900 reactions.

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

What volume will I receive with the Custom and Custom Plus TaqMan RNA assays for the different scales?

The Custom and Custom Plus TaqMan RNA assays are supplied with the following volumes:
Small: 360 µL
Medium: 750 µL
Large: 976 µL

Please note that the large scale is supplied in a 60X format, while the small and medium scales are supplied in 20X format.

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

What formats are available for Custom and Custom Plus TaqMan RNA assays?

Custom and Custom Plus TaqMan RNA assays can be ordered online with either FAM, VIC, or VIC primer limited.

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

Which reporter dyes and quenchers are available for Custom TaqMan RNA assays and Custom Plus TaqMan RNA assays?

Custom and Custom Plus TaqMan RNA assays can be ordered online with either FAM or VIC dye, with a MGB-NFQ quencher.

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

What reporter dyes are available for the 5' labeling of custom TaqMan probes?

Fluorescent dyes for the 5' labeling of TaqMan MGB probes and recommend for use with Applied Biosystem instruments are: 6-FAM, VIC, ABY (NED for the StepOnePlus), JUN and Cy5.

For TaqMan TAMRA probes, recommended dyes are: 6-FAM and VIC.

Be sure to check the specifications for each instrument to confirm available filters and compatible dyes. More detailed information can be found at: https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/taqman-custom-plating-manufacturing/taqman-custom-assay-oligo.html