Why had my RT² SYBR Green Mastermix been working well in the past, but now does not seem to be?

The sudden failure of a re-used RT² SYBR Green Mastermix is most likely due to repeated warming of the same 200-reaction scale product. We do not recommend repeatedly removing the same vial of PCR master mix from the -20 ºC freezer. The enzyme activity will decrease over time, under these conditions. Instead, upon receiving the master mix, divide into aliquots of a volume that you predict you will use for each day's experiment. Any unused portion of an aliquot should either be discarded or saved, noting that it has been used previously, for less critical uses or experiments. Make sure that you do not store the RT² SYBR Green Mastermixes frozen at -80 ºC, as this will kill master mix activity.