Fluorophores and Quenchers

Dual-labeled probes (also called hydrolyzation probes or TaqMan® probes) are usually labeled with the reporter at the end 5´ and with the quencher placed at the end 3´. Choice of the reporter depends on excitation and emission channels of the used real-time PCR cycler (almost all real-time PCR cyclers provide a channel for fluorescein - FAM).

Today, BHQ (Black Hole Quencher) type quenchers are usually used as quenchers; they quench a broad spectrum of fluorophores and show no autofluorescence. For more detailed information regarding the use and interplay of the most common fluorophores/ reporters and quenchers, please download the flyer “Fluorophores and Quenchers”.

To learn more about our prices, please login to our webshop and download the current price list. Microsynth supplies hydrolyzation probes which are specified in more detail in the table below.

Product Characteristics:

5‘ Label	3‘ Label	0.2 µmol scale1		1.0 µmol scale1
		Guaranteed yield2	Average yield2	Guaranteed yield2	Average yield2
FAM HEX TET	TAMRA	2 OD260 10 nmol	8 OD260 40 nmol	10 OD260 50 nmol	30 OD260 150 nmol
FAM HEX TET	QSY-7	1.5 OD260 7.5 nmol	5 OD260 25 nmol	8 OD 40 nmol	15 OD 75 nmol
AlexaFluor 350 FAM HEX TET	Dabcyl	2 OD260 10 nmol	5 OD260 25 nmol	10 OD260 50 nmol	20 OD260 100 nmol
AlexaFluor 350 FAM HEX JOE TET ATTO 425 / 532 Yakima Yellow (equivalent to VIC)	BHQ-1	1.5 OD260 7.5 nmol	6 OD260 30 nmol	8 OD2603 40 nmol3	25 OD2603 125 nmol3
ATTO 647N / 620 ATTO 550 (equivalent to NED) Cy3 / Cy5 Dyomics 681 ROX TAMRA Texas Red	BHQ-2	1.5 OD260 7.5 nmol	4 OD260 20 nmol	8 OD 40 nmol	15 OD 75 nmol
For 1.0 µmol synthesis scales with no indication, please contact us for pricing. If your desired fluorophore or quencher is not listed in this table, please contact us for availability and pricing.

Potential Applications:

• Gene expression
• Multiplex assay development
• Pathogen detection
• Viral load quantitation
• Microarray validation
• si/shRNA knockdown
• Gene copy determination
• Mutation detection
• Allelic discrimination
• SNP detection

Some Remarks to the Correlation of Yield and Number of Reactions

In the case of real-time PCR, usually one measurement or reaction requires 20 µl of a solution containing the probe at a concentration of 0.2 pmol/µl. As a consequence, one reaction consumes 4 pmol of a probe. For instance, if you order a 5’ FAM + 3’ TAMRA probe at 0.2 µmol synthesis scale we deliver at least 10 nmol of the specified probe to you. Therefore, you can theoretically perform at least 2’500 reactions (whereas the average delivered amount of ~40 nmol is sufficient for ~10'000 reactions).

Calculation of the number of reactions for real-time PCR (quantity of probe: 4 pmol):

# reactions = delivered amount [nmol] * 1’000 / 4

 

How to Order?

• Enter our webshop
• Click on DNA and metRNA in the "Oligonucleotide" domain
• Select either Normal Entry in order to type or copy/paste the desired sequence information etc. or alternatively select Upload Entry by using our convenient Excel Template (can be downloaded during ordering)
• Simply select your desired 5' and 3' modification, in this way your oligo is recognized and processed as a TaqMan probe (including two-step HPLC or PAGE purification).

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¹ The synthesis scale represents the initial amount of 3' bases (starting material).

² Yields indicated in OD260 apply to a probe consisting of 20 DNA bases; Calculation: 1 OD = 5 nmol (please note that this calculation is based on sequences with virtually homogenous distribution of the 4 DNA bases; it may vary for sequences with high GC contents >70% etc.)

³ guaranteed and average yields indicated are only valid for 5’ labels with FAM, HEX, JOE and ATTO 425 / 532