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Fluorophores and Quenchers

Microsynth offers a wide range of fluorophores and quenchers in various combinations that are suitable for each used method and real-time thermocycler. Traditionally, FAM-TAMRA is one of the most frequently used pairs for TaqMan probes, where FAM acts as the fluorophore and TAMRA as the quencher. Also other quenchers can be used, especially dark quenchers or black hole quenchers which capture energy from an excited reporter molecule without subsequent emission of light, i.e. they do not fluorescence.

Probes made by using dark quenchers tend to be more sensitive in quantitative detection systems, primarily due to lower background fluorescence and a better signal to noise ratio than probes that contain fluorescent quenchers. Moreover dark quenchers enable the use of a wider range of reporter dyes, expanding the options available for multiplexed or genotyping assays.

Some fluorophores can be quenched by more than one quencher. In any case the absorption spectrum of the quencher needs to have a good overlap with the emission spectrum of the fluorophore to achieve optimal quenching. Quenchers have a quenching capacity throughout their absorption spectrum, but the performance is best close to the absorption maximum.

 

For multiplex PCR we recommend the following 5’fluorophore 3’-black hole quencher combination for most qPCR thermo cyclers using TaqMan probes:

Channel 1: FAM-BHQ1
Channel 2: Yakima Yellow*-BHQ1 (*equivalent to VIC)
Channel 3: ATTO550**-BHQ2 (**equivalent to NED)
Channel 4: ROX-BHQ2

Recommended final concentrations for standard experiments:
Primers: 0.9 pmol/ul (0.9 uM)
Probe: 0.2 pmol/ul (0.2 uM)

Recommended RT-PCR reaction (Tm Primer: 59 °C, Tm Probe: 69 °C):
30 sec 95 °C
30 sec 57 °C
30 sec 72 °C, 35 cycles

Schematic of fluorophores and their corresponding quenchers, which can be ordered at Microsynth. (just click on the graphic to open a PDF-file)


5’ Label

3’ Label

Synthesis scale

Guaranteed Yield

Average Yield

Product number

FAM

HEX

TET

 

TAMRA

 

0.2 µmol

 

2 OD260

 

7 OD260

0113

0117

0118

FAM

HEX

TET

 

TAMRA

 

1.0 µmol

 

10 OD260

 

30 OD260

0160

0164

0165

FAM

HEX

TET

 

QSY-7

 

0.2 µmol

 

1.5 OD260

 

2 OD260

0131

0132

0133

AlexaFluor 350 

FAM

HEX

TET

Dabcyl

0.2 µmol

2 OD260

5 OD260

0125

0115

0119

0121

AlexaFluor 350

FAM

HEX

JOE

TET

ATTO 532

YAKIMA YELLOW

(equivalent to VIC)

 

BHQ-1

 

0.2 µmol

 

2 OD260

 

6 OD260

0126

0169

0170

0175

0171

0127

0172

FAM

HEX

JOE

TET

YAKIMA YELLOW

(equivalent to VIC)

BHQ-1

1.0 µmol

10 OD260

25 OD260

0179

0183

0182

0184

0178

ATTO 647N

ATTO 550

(equivalent to NED)

ATTO 620

CY3

CY5

Cy5.5

Dyomics 681

ROX

TAMRA

BHQ-2

0.2 µmol

1.5 OD260

3 OD260

0145

0140

 

0141

0173

0174

0143

0142

0177

0176

 

For other ATTO-, Dyomics-, and Alexa Fluor-Modifier, please inquire.

For other probes in the 1 µmol synthesis scale, please inquire.

 
www.microsynth.ch
www.microsynth.ch