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General

A TaqMan® probe is a single DNA oligo, containing a 5’ reporter dye and a 3’ quencher. A TaqMan® Assay is a single tube containing a forward primer, a TaqMan® probe, and reverse primer premixed together at specified concentrations.

TaqMan® MGB probes have the following features: 

1) A fluorescent reporter at the 5’ end (typically FAM™ or VIC® dye)
2) A nonfluorescent quencher at the 3’ end. Because the quencher does not fluoresce, the real-time PCR 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. 

Note: A TAMRA™ quencher does not affect the Tm of the probe.

In general, the TaqMan® MGB probes exhibit great differences in Tm values between matched and mismatched probes, which provides more accurate allelic discrimination and a more sensitive real-time assay. Mismatches between a probe and target reduce the efficiency of probe hybridization in a measurable way, which is especially important in SNP genotyping assays.

A QSY® quencher is also a nonfluorescent quencher and is available with a variety of dyes to support multiplexing. Similar to MGB, because the quencher does not fluoresce, the real-time instruments can measure the reporter dye contributions more precisely.

A TAMRA™ quencher is a weakly fluorescent quencher. You must keep this in mind if you plan to multiplex with such a probe, as the TAMRA™ quencher will take up a dye option on your instrument.

When choosing a quencher, keep in mind the basic types of quenchers. They can be nonfluorescent or weakly fluorescent; they can increase the Tm of the probe or have no effect on the Tm. If you plan to multiplex, using a nonfluorescent quencher is usually best.

The choice of quencher will depend on whether you are designing a probe from scratch or you already have a sequence (a probe sequence from a publication, for example). If you are designing from scratch, you will have the flexibility to alter the sequence to meet Tm requirements as needed. In general we prefer MGB-NFQ as a quencher, as it allows for shorter probes which are more specific. If you are interested in multiplexing, we recommend the QSY® probe. If you are using a published sequence, you will want to use the same quencher, or if it is not available, then the same type of quencher. However, if you would like to alter the sequence to change a fluorescent quencher to an NFQ-MGB quencher, you can use our Primer Express® Software.

We do not have BHQ® as a quencher option for our probes, but we do offer QSY® quencher as an alternative. Using QSY® quencher requires no redesign of the probe, so it is a simple change from one to the other. We also offer MGB-NFQ as an alternative nonfluorescent quencher, but a redesign of your probe would be necessary since the Tm of a BHQ® probe and an MGB-NFQ probe will not be the same. (MGB increases the Tm of the probe.)

Below is a table of our instruments and the recommended dye combinations for multiplexing:

Instrument

Number of Filters and Dyes Available

Passive Reference Dye

Dyes Available for Multiplexing

StepOne™

3

ROX™

FAM™

VIC™

 

 

StepOnePlus™

4

ROX™

FAM™

VIC™

NED™

 

7300

4

ROX™

FAM™

VIC™

NED™

 

7900HT Fast

4

ROX™

FAM™

VIC™

NED™

 

7500/7500 Fast

5

ROX™*

FAM™

VIC™

NED™ or ABY™

JUN™

ViiA™ 7

6

ROX™*

FAM™

VIC™

NED™ or ABY™

JUN™

QuantStudio™ 12K Flex

6

ROX™*

FAM™

VIC™

NED™ or ABY™

JUN™

QuantStudio™ 6 Flex

5

ROX™*

FAM™

VIC™

NED™ or ABY™

JUN™

QuantStudio™ 7 Flex

6

ROX™*

FAM™

VIC™

NED™ or ABY™

JUN™

QuantStudio™ 3

4

ROX™*

FAM™

VIC™

NED™ or ABY™

JUN™

QuantStudio™ 5 (96 well block)

6

ROX™*

FAM™

VIC™

NED™ or ABY™

JUN™

QuantStudio™ 5 (384 well block)

5

ROX™*

FAM™

VIC™

NED™ or ABY™

JUN™

*Note: If JUN® is used as a custom probe, MUSTANG PURPLE™ dye should be used instead of ROX™ dye for the passive reference dye.

All custom MGB probes are shipped resuspended at 100 µM (100 pmol/µL) in 1X TE. Custom QSY probes and TAMRA™ probes can be shipped either resuspended at 100 µM (100 pmol/µL) in 1X TE or dried down. The concentration should be on your tube label as well.

This means that you should receive the following volume based on what you ordered for custom MGB probes:

6,000 pmol order = 60 µL of custom probe in 1X TE (100 µM)
20,000 pmol order = 200 µL of custom probe in 1X TE (100 µM)
50,000 pmol order = 500 µL of custom probe in 1X TE (100 µM)

Storage

Custom primers and probes are good for at least 12 months from the date of receipt if stored properly.

We recommend diluting the TaqMan® Assays with 1X TE buffer. (Note: The 1X TE buffer should be 10 mM Tris-HCl, 1 mM EDTA, pH 8.0, and be made using DNase-free, sterile-filtered water.)

Once the assay or probe is reconstituted and/or diluted, it is recommended that it be aliquoted into smaller volumes and then stored at –20°C. Making small aliquots reduces the number of freeze-thaw cycles and therefore will extend their shelf life. We recommended limiting the number of freeze-thaw cycles to no more than 10.

Here are the estimated number of reactions you would get out of a custom probe or primer, assuming a final probe concentration of 250 nM, or primer concentration of 900 nM.

Custom probe size

Number of reactions (at 20 µL final reaction volume)

Number of reactions (at 10 µL final reaction volume)

6,000 pmol

1,200

2,400

20,000 pmol

4,000

8,000

50,000 pmol

10,000

20,000

Custom primer size

Number of reactions (at 20 µL final reaction volume)

Number of reactions (at 10 µL final reaction volume)

10,000 pmol

555

1,111

80,000 pmol

4,444

8,888

130,000 pmol

7,222

14,444

Ordering

You can search for predesigned TaqMan® assays here. Please refer to this handy video for more guidance on how to select a TaqMan® Gene Expression Assay.

Yes. If your search does not turn up any results, simply click on the option for “Design a Custom Assay” that appears towards the top of the page. This will take you to the appropriate Custom Assay Design Tool in which you can submit your sequence for a custom design.

If you would like a reporter dye other than 6-FAM™ dye, or would like a different formulation of an existing predesigned TaqMan® assay, you can order it here by providing the custom assay ID.

You can order a TaqMan® probe with 6-FAM™, VIC®, NED™, JUN®, ABY®, or TET™ dye here.

Yes. You can order a TaqMan® probe with NED™, JUN®, ABY®, or TET™ dye here. If you would like a reporter dye other than those above, or would like a different formulation of an existing predesigned TaqMan® assay, you can order it here .

Custom probes typically take 5–12 business days to arrive once ordered.

Yes. For a gene expression assay, you can order here, by selecting the “Enter Custom Primer/Probe Pairs” option. For SNP genotyping assays, you can order here by selecting the “Enter Custom Primer Probe Pairs” option.