To demonstrate the validity of Transcriptic’s Autoprotocol-implemented Plate Reading Quick Instruction.
Assessment of plate reading measurements employing multiple wavelengths between 475 - 535 nm was achieved using two independent experiments, performed in the same measurement plate (96-well, flat-bottom, optically-clear). Measurements were recorded using Infinite 200 Pro (Tecan) instrumentation. Utilizing dye with known absorption and a SYBR® Green-based DNA quantification assay, both absorbance and fluorescent measurement was assessed.
Orange G electrophoresis dye (CHEM IMPEX) solutions of 100, 50, 25 and 12.5 µg/ml were prepared in distilled water. One-hundred microliters of dye were pipetted into rows A, B, C and D of a 96-well plate respectively (Figure 1). Absorbance measurements (475 nm) of rows A-D were performed simultaneously using the Plate Reading Quick Instruction.
Ninety-nine microliters of SYBR® Green 1 (Life Technologies- diluted 1:2000 in TE), which fluoresces in the presence of dsDNA, was pipetted into rows E-H of a 96-well plate. One-microliter of 50, 25,12.5 and 6.25 ng/µl DNA (derived from a 100 ng/µl lambda DNA standard, Promega) dissolved in TE, was added to rows E-H respectively (Figure 1). Fluorescence measurements (exc. 485 nm, em. 535 nm) of rows E-H were performed simultaneously using the Plate Reading Quick Instruction.
The Plate Reading Quick Instruction achieved consistent absorbance and fluorescence readings with minimal deviation observed across replicates. Observed readings were consistent with anticipated linear relationships (dye concentration vs. absorbance; DNA concentration vs. fluorescence).
VALIDATION DATE: September 2015