Agilent eBook - 8

Best Practices with an Agilent Fragment Analyzer System

of the sample significantly and resulted in a tight
concentration range (0.936 to 1.078 ng/µL), a low
standard deviation, and an excellent precision (low % CV).
Three more rows were prepared with the same sample
and diluent marker. Each row was mixed 10 times
according to the suggested mixing protocol listed in
the kit manuals: swirling while pipetting up and down
at 2 µL volume, swirling while pipetting up and down at
20 µL volume, and electronic pipetting at 10 µL volume
(Table 2). All reported accuracies were under 5 % error,
with precision values under 6 % CV. Mixing less than
10 times resulted in higher % error and lower precision
with the 2 µL volume (data not shown). To avoid variation
from mixing techniques, a plate vortexer/shaker is the
recommended method for mixing. All recommended
mixing methods resulted in quantification precision
and accuracy values within the specifications of the
Agilent HS NGS Fragment kit.
Eliminating pipetting errors

Pipetting introduces intrinsic error into sample
handling. It is crucial that pipettes are calibrated,
and the proper pipette is used for each volume. For
instance, a 2.5 µL pipette would be more accurate for
a 1 µL sample than a 20 µL pipette. Appropriate fitting
pipette tips also plays a role in eliminating pipetting
errors. Proper pipetting techniques ensure excellent
precision and consistent quantification. If analyzing
replicates, a master mix of the sample and diluent



Table 2. Lack of mixing greatly effects precision, accuracy, and individual sample quantification as seen with a high standard deviation and % CV.
Several mixing methods are suggested in the kit manuals. All mixing methods produce similar precision and accuracy if completed a minimum of
10 times. Samples were separated on the Agilent 5200 Fragment Analyzer system with the Agilent HS NGS Fragment kit (1‑6000 bp) a: n=6; b: n=12.
A master mix provides the most consistent quantification as seen by % error.

No mixingb

2 µL Pipette mixing

20 µL Pipette mixing

10 µL Electronic
pipette mixing 10×a

Plate shakerb

Master mix







0.17 to 0.81

0.99 to 1.13

0.99 to 1.02

0.92 to 1.04

0.936 to 1.08

0.99 to 1.09







36.9 %

5.5 %

2.3 %

5.0 %

4.7 %

3.5 %

44 %

4.2 %

3.5 %

4.1 %

3.3 %

1.7 %

Average (ng/µL)
Range (ng/µL)
Standard deviation
Precision % CV
Accuracy % error

marker is recommended to achieve the best
quantification precision and accuracy. A master mix
involves adding the correct ratio of sample and diluent
marker for an entire row to a low-bind tube, followed
by vortexing, and aliquoting the mixture into each well.
This eliminates error from individually pipetting the
sample and diluent marker into each well. As seen in
Figure 1C, consistent quantification is achieved across
the entire row. The master mix demonstrated baseline
reproducibility and provided even better precision and
accuracy by eliminating variation from individual
pipetting (Table 2).
Nanodrop versus Qubit

Quantification on the Agilent 5200 Fragment Analyzer
system is often compared to the Qubit fluorometer

or the Nanodrop spectrophotometer. Both the Agilent
5200 Fragment Analyzer system and the Qubit utilize
fluorescence detection, while the Nanodrop uses
UV-Vis spectrophotometry.
Several factors need to be considered when assessing
nucleic acid concentration with UV-Vis spectrophotometry.
Nucleic acids such as RNA, ssDNA, and dsDNA all
absorb at 260 nm. Contamination sources can absorb
at the same wavelengths as nucleic acids. Protein, a
common contamination source, can artificially increase
DNA concentration through overlap at 260 nm from the
280 nm peak. In addition, proteins with high levels of
the amino acids phenylalanine and histidine, or proteins
with certain metals bound to them, are more likely to
absorb around 260 nm. Furthermore, detergents with

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