plate reading

EXPERIMENTAL OBJECTIVES

To demonstrate the validity of Transcriptic’s Autoprotocol-implemented Plate Reading Quick Instruction.

methodology

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.

Figure 1  A single plate was used for both absorbance and fluorescence readings, performed by two independent sequential Plate Reading Quick Instructions.  Rows A-E, filled with diminishing concentrations of Orange G, were assayed for absorbance at 475 nm.  Rows E-H, filled with samples of diminishing DNA concentration with a constant concentration of SYBR® Green 1 dye (which fluoresces in the presence of dsDNA), were assayed for fluorescence (exc. 485 nm, em. 535 nm).

Figure 1  A single plate was used for both absorbance and fluorescence readings, performed by two independent sequential Plate Reading Quick Instructions.  Rows A-E, filled with diminishing concentrations of Orange G, were assayed for absorbance at 475 nm.  Rows E-H, filled with samples of diminishing DNA concentration with a constant concentration of SYBR® Green 1 dye (which fluoresces in the presence of dsDNA), were assayed for fluorescence (exc. 485 nm, em. 535 nm).

Results

Figure 2  (A) Heat map representation of absorbance and fluorescence data obtained from 96-wells.  The heat map is a composite of data obtained from independent absorbance (experiment 1, blue gradient) and a fluorescence assessments (experiment 2, gray gradient) of the same plate.  (B) Statistical analysis of all absorbance/fluorescent readings. C.V.: coefficient of variance.  R.F.U.: relative fluorescence units.  (C) Graphical representation of absorbance readings.  Each point represents the mean absorbance of twelve replicate (each row) samples of distinct Orange G concentration.  Error bars: standard deviation.  Trend line: linear.  (D) Graphical representation of fluorescence readings.  Each point represents the mean absorbance of twelve replicate (each row) samples of distinct DNA concentration.  Error bars: standard deviation.  Trend line: log10.

Figure 2  (A) Heat map representation of absorbance and fluorescence data obtained from 96-wells.  The heat map is a composite of data obtained from independent absorbance (experiment 1, blue gradient) and a fluorescence assessments (experiment 2, gray gradient) of the same plate.  (B) Statistical analysis of all absorbance/fluorescent readings. C.V.: coefficient of variance.  R.F.U.: relative fluorescence units.  (C) Graphical representation of absorbance readings.  Each point represents the mean absorbance of twelve replicate (each row) samples of distinct Orange G concentration.  Error bars: standard deviation.  Trend line: linear.  (D) Graphical representation of fluorescence readings.  Each point represents the mean absorbance of twelve replicate (each row) samples of distinct DNA concentration.  Error bars: standard deviation.  Trend line: log10.

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