Dear researchers,
I plan to do a duplex qPCR with two primers and each primer has a probe. First one uses Cy5 as fluorophore and BHQ-1 as quencher and the second one uses FAM as fluorophore and BHQ-1 as quencher. In our lab, we have a Mastercycler realplex4 which doesn't have Cy5 set as dyes. What fluorophore can I use instead? Both probes have to have BHQ-1 as quencers?
Thank you very much for helping a less experienced fellow.
Dear Roxana, BHQ-1 is not an ideal candidate for Cy3 and Cy5 series dyes. BHQ-1 is best to use with fluorophores such as FAM, TAMRA, ROX, VIC, JOE etc. For Cy5 level dyes it is best to adopt BHQ-2 quencher. Anyway it will work for Cy5 but not being the best suited one. Also, the best alternate of Cy5 is TYE 665 only. Although, APC and AF 647 are some other alternatives for Cy5, but they are mainly used in Flow Cytometry.
Regards....
Hi,
just checked the document at Eurofins and based on this I would either go with Hex or Rox labelled probes. In principle it is not the biggest challenge to swab the fluorophores you just need to check, that you do not have a crosstalk between the channels (would recommend to run the assays singleplex and duplex on one plate and check if you get a unwanted signal for the singleplex assay in the other channel). BHQ-1 works good for Hex, for ROX I woud go for BHQ-2
Good look
Sven
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