Bio-Rad Experion DNA Analysis Kits - Manual (Page 52)

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Fig . A .3 . Results tab generated by an Experion DNA 1K analysis . Shown is the Results tab for a single sample.
Data Analysis: Normalization, Sizing, and Quantitation
Following separation, Experion software analyses DNA fragments using one or more of the following:
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Internal markers (upper and lower) for normalizing migration times of samples in different
wells
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DNA ladder for determining fragment size (sizing)
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Internal upper marker for determining concentration (quantitation)
Normalization: Aligning Peak Migration Times
To compensate for small variations in each separation, Experion DNA analysis uses two internal
markers to normalize the migration times among samples. The two internal markers, an upper marker
(1,500 bp/17,000 bp) and lower marker (15 bp/50 bp), are included in the Experion DNA loading buffer.
Therefore, both of these markers are added to each sample and the DNA ladder (Figure A.1). Inclusion
of these markers and the normalization process ensure that the software properly identifies peaks.
DNA Sizing: Determining Peak Sizes
The first sample to be analyzed is the DNA ladder, which contains 13 DNA fragments (including two
markers) of 15–1,500 bp (DNA 1K) or 50–17,000 bp (DNA 12K). Experion software constructs a standard
curve of migration time as a function of fragment size from the ladder separation. It then calculates sizes
of the DNA fragments from the sample wells by comparing their migration times to the standard curve.
Quantitation: Determining DNA Amount
Experion software offers two different types of DNA quantitation methods: percentage determination
and concentration determination. All quantitation measurements are based on the time-corrected peak
area (corrected area) of each peak identified in an electropherogram. The corrected area of a peak is
proportional to the amount of the DNA it represents in a mixture.
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Percentage determination — measures the percentage of each fragment in a mixture
(% total). This method is commonly used for determining DNA content, purity, and stability.
The accuracy of this method depends on the dye-binding efficiency of each component in
the DNA mixture
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Concentration determination — provides the amount of the fragment(s) in a mixture
rather than just a percentage of the total. Experion software quantitates a DNA sample
peak by comparing the sample peak area relative to the concentration:peak area ratio of
the upper marker and applying a correction factor to account for the size dependence of
fluorescence
Experion Automated Electrophoresis System