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?

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Answer

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.

Answer Id: E2740

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4a6a3a1c58a29944c3d8dd30701d5652_FAQ

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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c6fa0858af353629b94e322efb5117f3_FAQ

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

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Answer

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.

Answer Id: E1371

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bd9ac49d36e1a373a4d3d94200ceafd7_FAQ

How can I calculate the concentration of a TaqMan® probe?

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Answer

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.

Answer Id: E1512

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b5ec3ff028d7c678e9694eb749d1579e_FAQ

Can I replace a TaqMan® probe with fluorescent primers?

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Answer

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.

Answer Id: E1575

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327f6e0ab05e96e629ba0952d8492abf_FAQ

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

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Answer

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.

Answer Id: E1576

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1c5d220b8a71718eef2243b0e7ec552a_FAQ

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?

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Answer

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.

Answer Id: E1772

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