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
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.
