Detection of PCR products in real time using light-up probes.

tometric procedures to within an average of 5% (CS) or 10% (CPT) and with the NADH fluorometric procedure for CS to within an average of 1% (Table 2). Precision, as indicated by the coefficient of variation (CV) for sample replicates, averaged 4.4% for the CS ABD-F assays and 1.7 and 2.3% for NADH fluorometric and spectrophotometric assays, respectively. CVs for CPT are 6% for both procedures and are similar to those reported for an alternative fluorometric assay (14). Scaling down ABD-F assay. If necessary, assay volumes may be scaled down. When assays are conducted on samples with relatively high enzymatic activities (as was the case for CS in the present work), the protein precipitation step and subsequent centrifugation may be omitted (i.e., the small contribution of protein thiols to blank fluorescence may be tolerated). In such cases, the assay may be adapted for highthroughput microwell plates with reagent volumes reduced appropriately. For example, in measuring CS activity in individual copepods (Class Crustacea) weighing approximately 100–200 mg dry weight, we have modified the protocol to conduct the assay in a 96-well microplate using 100 ml reagent medium and 10 ml homogenate (one copepod in 500 ml homogenizing medium). The reaction is stopped with the addition of 20 ml SSA (17.5%), neutralized with 50 ml of KHCO3 (1 M) before labeling with 20 ml of ABD-F (2 mg ml). KHCO3 is added slowly to avoid vigorous bubbling as CO2 is generated.