Agilent eBook - 12

Best Practices with an Agilent Parallel CE System

Experimental
The experiments in this study were done using a 5200 Fragment Analyzer system and
can be replicated with comparable results on Agilent 5300 and 5400 Fragment Analyzer
systems.
Various fragment sizes - 300, 1,000, and 15,000 bp - were analyzed on the 5200
Fragment Analyzer system with the Agilent HS NGS Fragment kit (1 to 6000 bp)
(p/n DNF-474) and the Agilent HS Large Fragment 50 kb kit (p/n DNF-464) over a
concentration range of 3.9 to 500 pg/µL and 4.5 to 600 pg/µL, respectively. A DNA
smear was separated with the Agilent HS Small Fragment kit (p/n DNF-477) over a
concentration range of 78 to 5,300 pg/µL. The Agilent HS NGS DNA Ladder (p/n DNF-396)
was diluted with 200, 100, 50, and 25 mM NaCl or 1× TE buffer and separated on the
5200 Fragment Analyzer system with the HS NGS Fragment kit.
Results and Discussion
Peak size versus smear size

Fragments and smears have different separation profiles on electropherograms.
Fragments are displayed as sharp peaks, while smears are broader and lower in height
than fragments with the same concentration. DNA libraries vary greatly in size and
profile, due to different modes of processing. They often have profiles similar to smears.
However, libraries with a small size range can display a profile characteristic of both
fragments and smears.
DNA size can be reported as a peak size or smear size. The peak size is the tallest/most
concentrated portion of the sample, while the smear size accounts for the entire distribution
of the sample over the designated smear range. ProSize automatically reports a peak size
for all sample types. The Smear Analysis tab in ProSize allows the user to set a base pair
range for determination of the average size and concentration of the smear. Peak size
is recommended for sizing of DNA fragments.

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