Hi, I want to add an additional dye to my already functioning multiplex taqman QPCR. the two probes I already am using are 6FAM-TAM and Joe-TAM What dyes would be appropriate for a 3.rd probe? iam using an applied biosystems machine (either Viia7 or StepOne plus) thank you all.
Kenneth, you can try add probe with ROX-BHQ2 or Cy5-BHQ2(or BHQ3). They have spectra far from FAM and Joe (and also TAMRA). If it possible, you should use probes FAM-BHQ and Joe-BHQ instead of TAMRA as a quencher (you take less background with BHQ).
Good luck!
Hi, you may choose appropriate dyes that are compatible to your machine/filter set.
For example, you can't use ROX because in applied biosystem machines it is used as passive reference dye. For StepOnePlus only few dyes are readed (FAM, TET, HEX, JOE, Tamra), so considering that you already have FAM (~520 nm) and JOE (~550 nm) you can choose TET (~536 nm). The quenchers BHQ are better for multiplexing, furthermore you can useTamra as a reporter dye (his emission is at 583 nm) with BHQ2 as quencher.
I have now ordered TAM-BHQ2 as the 3.rd duallbled probe. Thank you..
Hi Kennet,
I have a very similar situation,
Can you tell me how it worked for you?
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