picogreen assay

EXPERIMENTAL objectiveS

To demonstrate the validity of Transcriptic’s Autoprotocol-implemented DNA Quantification protocol.

methodology

Using the DNA Quantification Protocol, a standard curve of DNA samples was generated.  SYBR® Green 1 dye (Life Technologies) fluorescence was used as a read-out for the concentration of dsDNA (Lambda DNA, New England Biolabs) serially diluted in TE to create an eight-point standard curve (50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39 ng/µl).  Each standard was pipetted in triplicate by combining 99 µl SYBR® Green 1 dye (diluted 1:2000 in TE) with 1 µl DNA solution (in TE) and fluorescence (exc. 485 nm, em. 535 nm) of each replicate measured using Infinite Pro 200 (Tecan) instrumentation.

Results

The DNA Quantification Protocol successfully generated a standard curve using standards of known DNA concentration (0-50 ng/µl).  Plotting of log10[DNA concentration] vs. fluorescence generated a linear trend. 

Figure 1  (A) Relative fluorescence units (RFU) plotted against DNA concentration.  Each point represents the mean RFU derived from triplicate measurements.  Error bars: standard deviation (S.D.).  Trend line: log10.  (B) Relative fluorescence units (RFU) plotted against log10[DNA concentration].  Each point represents mean RFU derived from triplicate measurements.  Error bars: standard deviation (S.D.).  Trend line: linear.  (C) Raw fluorescent data obtained. C.V.: coefficient of variance.

Figure 1  (A) Relative fluorescence units (RFU) plotted against DNA concentration.  Each point represents the mean RFU derived from triplicate measurements.  Error bars: standard deviation (S.D.).  Trend line: log10.  (B) Relative fluorescence units (RFU) plotted against log10[DNA concentration].  Each point represents mean RFU derived from triplicate measurements.  Error bars: standard deviation (S.D.).  Trend line: linear.  (C) Raw fluorescent data obtained. C.V.: coefficient of variance.

 

VALIDATION DATE:  September 2015