Abstract: A limited well thermal cycling device for sample preparation and real-time fluorescence detection is described. The limited well thermal cycling device includes a heating block having a sample well and at least one reaction well and an optical block including a corresponding means for measuring real time fluorescence in each reaction well. The limited well thermal cycling device includes a means for efficient heating and cooling of reaction and sample wells for real-time fluorescence detection. The structure of the heating block provides the means for efficient heating and cooling by having each of the sample well and at least one reaction wells rising above the heating block base, such that the sample well and at least one reaction well are not surrounded by the metal heating block.