Agilent eBook - 9

Best Practices with an Agilent Parallel CE System

aromatic groups can also absorb in the same region increasing
the apparent total concentration of the DNA in the sample.
Fluorescence detection of nucleic acids utilizes a dye that
fluoresces only when bound to nucleic acids. Unbound dye does
not fluoresce, eliminating unwanted background interference.
Fluorescence dyes, in general, have a high specificity for their
target molecule. Thus, interference from contaminants such as
free proteins and carbohydrates are not an issue when determining
nucleic acids concentration. Sheared DNA and human gDNA
concentrations were compared on the Nanodrop, Qubit, and
Agilent 5200 Fragment Analyzer system (Table 3). Sheared
DNA samples (1 to 4) were separated with the Agilent HS NGS
Fragment kit (1-6000 bp). The human gDNA samples (5 to 7)
were separated with the Agilent Genomic DNA kit. The
Nanodrop consistently reported a higher concentration
compared to the Qubit and Agilent 5200 Fragment Analyzer
system. If comparison of DNA sample concentrations is
necessary, we recommend comparing quantification between
the Agilent 5200 Fragment Analyzer system and the Qubit
due to the use of the same detection method.
Effects of salt

Awareness of the salt concentration in samples is important for
best quantitative results. The Agilent 5200 Fragment Analyzer
system sample preparation protocol recommends that all
samples are diluted with 1× TE buffer (10 mM Tris-HCl, 1 mM
EDTA) and the chloride salt concentration in the sample must
remain below 10 mM. High salt concentrations may cause
noisy baselines, sporadic spikes in the electropherogram, and

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Table 3. Quantification comparison between the Nanodrop, Qubit, and Agilent 5200 Fragment Analyzer system. The Agilent 5200 Fragment
Analyzer system and Qubit both utilize fluorescence for detection. The Qubit is recommended for quantification comparisons with the
Agilent 5200 Fragment Analyzer system due to the use of the same detection method. Samples 1 to 4 were sheared DNA separated with
the Agilent HS NGS Fragment kit (1‑6000 bp) and samples 5 to 7 were human gDNA separated with the Agilent Genomic DNA kit. a: n=2; b:
n=3; c: n=18.

Nanodrop (UV-Vis)

Qubit (fluorescence)

5200 Fragment Analyzer system
(fluorescence)

Concentration (ng/µL)

Avg.

% CV

Avg.

% CV

Avg.

% CV

6.15a

3.4 %

2.19b

3.4 %

2.21b

5.9 %

5.55a

3.8 %

3.07b

3.3 %

2.37b

1.8 %

5.1a

0.0 %

3.30b

1.6 %

3.22b

6.9 %

5.2a

5.4 %

3.59b

6.4 %

2.79b

5.8 %

197.6c

2.6 %

192c

2.3 %

181.9c

8.1 %

100.3c

2.1 %

95.9c

3.4 %

93.1c

7.6 %

49.2c

1.7 %

46.5c

4.6 %

43.9c

6.4 %

Sample
ID

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Agilent eBook

Table of Contents for the Digital Edition of Agilent eBook

Agilent eBook - 1
Agilent eBook - 2
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Agilent eBook - 25
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