my problem is my newly designed TaqMan FAM-TAMRA probes with beacon
designer are working quite strange on standard plasmid DNA in
monolplex runs. I get negative fluorescence signals duiring
monitoring of my reaction between -30,000 to -600,000 (raw data) while working with Eppendorf real plex4. I have used the system before with TaqMan-MGB probes for the same standard DNA and had never seen such strange runs. Secondly in all pairs of primers andf probes, pcr works in one or two of replicates out of three. I can not find reason of this out put and had never before something like this also. I dono if i need to optimize the reaction or could it be a bad synthesis of probe. As far as I know FAM-TAMRA chemisdtry works well in monoplex real time pcr.
urgent assistance is acknowledged.