Agilent eBook - 10

Best Practices with an Agilent Fragment Analyzer System

decreased quantification. The Agilent HS NGS DNA
Ladder was mixed with various concentrations of
NaCl (200, 100, 50, and 25 mM), separated with the
Agilent HS NGS Fragment kit, and demonstrated a
proportional decrease in sample concentration with
increased levels of salt (Figure 2). Reliable analysis
of DNA samples is ensured with the correct salt
concentration.
Input ladder concentration

ProSize uses the ladder concentration specified in
each kit manual to quantify the sample. The ladder
for each quantitative kit goes through rigorous
quality control checks to certify the concentration
with Qubit. However, quantification readings can
vary slightly between Qubit instruments due to the
surrounding environment, such as the temperature
of the lab. ProSize has the flexibility to adjust the
concentration of the ladder under the Quantification
tab. The ladder concentration can be replaced
with the users Qubit concentration measurement
in ProSize. Determining the concentration of the
Agilent 5200 Fragment Analyzer system ladder
and sample with the same Qubit, under the same
environmental conditions, will aid in achieving more
comparative quantification results between the
instruments.

HS NGS
Ladder

200 mM
NaCl

100 mM
NaCl

50 mM
NaCl

25 mM
NaCl

1× TE
buffer

6,000
3,000
2,000
1,500
1,000
800
700
600
500
400
300

200

100

1

Figure 2. Agilent HS DNA NGS Ladder analyzed on the Agilent 5200
Fragment Analyzer system with the Agilent HS NGS Fragment kit
(1‑6000 bp). Digital gel image of Agilent HS NGS DNA Ladder diluted in
200, 100, 50, and 25 mM NaCl or 1× TE (10 mM Tris‑HCl) buffer.

Evaporation

Evaporation of a sample over time is a concern,
especially during dry seasons and in drier climates.
Evaporation can decrease sample volume,
changing the dilution factor and affecting the
concentration of a sample. To avoid loss of sample
volume, a drop of mineral oil can be added to the
top of DNA samples in the 96-well plate. The oil
protects the samples from evaporation and retains
the ability to prepare and queue multiple runs for
unattended analysis.
Conclusions
The Agilent 5200 Fragment Analyzer system
provides automated analysis of DNA smears,
libraries, and fragments. Following the protocols
and best mixing practices described in each
kit manual and diluting the sample with the
correct buffer ensures reliable and accurate
DNA quantification. n
www.agilent.com
For Research Use Only. Not for use in
diagnostic procedures.
This information is subject to change
without notice.
© Agilent Technologies, Inc. 2019
5994-0513EN

10

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