Agilent eBook - 6

Best Practices with an Agilent Parallel CE System

Materials

The following kits were used together with the
Agilent 5200 Fragment Analyzer system: Agilent
HS NGS Fragment kit (1-6000 bp) (p/n DNF-474),
Agilent HS NGS DNA Ladder (p/n DNF-396-U100),
Agilent Genomic DNA kit (p/n DNF-487), and the
dsDNA HH kit (ThermoFisher Scientific, #Q32854).
A Qubit 2.0 fluorometer and a Nanodrop
spectrophotometer (both Thermo Fisher Scientific)
were used.
Results and discussion
Correct techniques
Agilent has developed qualitative kits specific for reliable
sizing and quality control, and quantitative kits explicitly
for accurate quantification, sizing, and quality control.
Each kit for the Agilent 5200 Fragment Analyzer system
is designed for a specific nucleic acid size and concentration range. It is important to choose the correct kit
based on the concentration and size of the sample to
achieve the best possible results. Most quantitative kits
have a standard sensitivity and high sensitivity version.
Generally, the standard sensitivity kits are for higher
concentration samples in the nanogram range, while
the high sensitivity (HS) kits are for lower concentration
samples in the picogram range.
It is important that the sample and ladder preparation
protocol is followed for each kit exactly. ProSize data

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Table 1. Recommended mixing protocols for various Agilent 5200 Fragment Analyzer system kits.

Swirl while pipetting
up and down
10× at 2 µL volume

Swirl while pipetting
up and down
10× at 20 µL volume

Plate shaker
3,000 rpm
for 2 min

Electronic pipettor 10×
at
10 µL volume

Small Fragment kit and HS Small Fragment kit

NGS Fragment kit and HS NGS Fragment kit
(1-6000 bp)

HS Large Fragment 50 kb kit
Large Fragment kit
HS Large Fragment kit

analysis software automatically loads a set of data
processing configurations specified for each kit method
and are tailored to the sample and ladder preparation
protocol described in the corresponding kit manual.
ProSize then calculates concentration based on preset
volumes of the sample, diluent marker, and ladder for
each kit. Any changes in the sample volume, diluent
marker volume, or ladder volume will affect the
quantification results provided by ProSize.
Bubbles are sometimes introduced to the 96-well
plate from preparation and mixing. Bubbles within
the sample can interfere with sample uptake into the
capillaries. It is advised with all kits to visually check
the plate and perform a quick spin to eliminate any
possibility of bubbles.
The use of low-bind tubes is recommended when
working with nucleic acids. This reduces the loss of

DNA or RNA caused by binding to the side of the tube
and ensures more accurate sampling. In addition, use
of the recommended 96-well plates ensures that the
capillary inlet is set at the proper level in the sample
matrix, aiding in reliable sample uptake by the capillaries,
since not all plate dimensions are the same.
Mixing
Each kit manual outlines a specific protocol for
proper mixing of the sample with the diluent marker
(Table 1). All Small Fragment, NGS Fragment, and
Large Fragment kit protocols add the diluent marker and
then the sample to the 96-well plate followed by mixing.
Mixing allows for homogenous distribution of the
sample throughout the well, enabling consistent
sample uptake, analysis, and accurate quantification.
Sample plates were prepared for analysis with the
Agilent HS NGS Fragment kit (1-6000 bp) to

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Agilent eBook

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Agilent eBook - 1
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