I optimized and got desired band with unlabeled primer set. But when i use labeled primer with HEX (Left primer labeled) i'm not able to get the band.
Three other labeled (other labels) primers worked fine with previously optimized conditions with unlabeled primer,
i never found that the dyes fam, tet or hex made any difference to getting a pcr band although in the analysis hex did give a weaker signal so a better product or lower dilution was needed to get the same signal strength. I would check very carefully that the sequence of the old unlabelled and the new labelled primers are identical, Also most pcr primers are just desalted so can be stored under many conditions but dye labelled primers are usually highly purified so need to be reconstituted in alkaline TE not water as water is acidic and can depurinate single stranded dna very easily. Is the template dna the same...it would be unfortunate to find that there is a polymorphism near the 3' end of one of the primers in your samples. try dropping your ennealing temperature ( a gradient would be ideal if your cycler allows it ) and double the amount of primer and include a control with the old primers added to check that the pcr is working
I found Hex work perfectly like FAM in my work. Please make sure you are using same thermal cycler with same PCR protocol when do comparison with labelled and non-labelled primers.
You should also consider contacting the company that made the primer for you. If you've followed the tips Paul mentioned and you still don't get a signal for your hex primer, it is possible they made a mistake and they should replace the primer free of charge. Are you checking your band on gel or on capillary? I have experienced fluorescent primers where the linking of the fluorophore to the primer was not done well, so no fluorescent signal was seen, but the band was there on gel.
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