BhQ2 is best, you should be guided by the emission peak (for dye) versus absorption peak for (quenchers)!
Yes, you can :)
You can take any reporter that is
1) efficiently quanched by the quancher (BHQs are quite promiscous) and
2) efficiently excited and detected by your intrument (depending on laser/LED wavelength and filters/detectors)
NED has the Excitation peak at 550nm, FAM at 495: check if FAM will be excited.
NED has the Emmission peak at 570nm, FAM at 515: check if FAM will be detected.
If both answers are "yes" then there is nothing to say against using FAM intead of NED. Most real-time cyclers should have the appropriate optics to measure FAM, so this shouldn't be a problem.
Note that Cy3 has the same spectral properties like NED (in case you are just looking for a possibly cheaper alternative). And instead of FAM you might consider HEX, JOE, VIC, or TMR, that have spectral properties that are more similar to NED than those of FAM.
Thanks for your quick answer. I have another question which quencher is the best for Cy3 BHQ-1 or BHQ-2 because there are different informations about this in articles
Hmm, difficult, since the Cy3 emmission peak is quite inbetween the absorption peaks of the two quenchers. The absorption peak of BHQ-2 is slightly higher, so I would expect a slightly more efficient energy transfer here, but this might also depend on the buffer conditions and the used linker. If you can, try both under your conditions and select the apparently better one. If you can't, I'd suggest to go for BHQ-2.
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