Improved Nucleic Acid QC with Parallel CE - 1

Application Note

Quantification Practices

Best Quantification Practices with the Fragment Analyzer™

The use of low-bind tubes is recommended when working with nucleic acids. This safeguards against 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
the capillary inlet is set at the proper level in the sample matrix for reliable sample uptake with the capillaries, since not all plate
dimensions are the same.

Abstract
Quality control checkpoints for concentration are a necessity for many nucleic acid applications and ensures successful outcomes.
The Fragment Analyzer™ Automated CE System from Advanced Analytical Technologies, Inc. (AATI) provides reliable quantification
for fragments and smears with PROSize® Data Analysis Software. This application note describes factors that affect quantification and
the best practices for achieving accurate quantification of the sample.

Mixing
Each kit manual outlines a specific protocol for proper mixing of the sample with the diluent marker (Table 1). The Small Fragment
(DNF-476, 477), NGS Fragment (DNF- 473, 474), and Large Fragment (DNF-464, 492, 493) Kit protocols all add the diluent marker and
then the sample to the 96-well plate followed by mixing.

Introduction

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 DNF-474 High Sensitivity NGS Fragment Analysis Kit (HS
NGS Fragment Kit) to demonstrate the effects of mixing on quantification (Figure 1). Standard deviation, precision, and

Accurate and reliable DNA quantification is essential for many applications such as PCR and library preparations. Reliable results
can be obtained when using best measurement practices on the Fragment Analyzer™ Automated CE System from Advanced
Analytical Technologies, Inc. (AATI). Best measurement practices can be optimized by choosing the correct analysis kit for the size
and concentration of the sample, thoroughly mixing the sample according to each kit protocol, minimizing pipetting error, and proper
sample preparation.

Concentration (ng/µL)

Results & Discussion
Proper Techniques
AATI 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 Fragment Analyzer 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 Kits are for lower concentration samples in the picogram
range.

Concentration (ng/µL)

It is important that the sample and ladder preparation protocol is followed for each kit exactly. PROSize® Data Analysis Software
automatically loads a set of data processing configurations specified for each kit method and 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.
Swirl while pipetting up &
down 10X at 20 µL volume

Plate Shaker
3,000 rpm 2 min

Small Fragment Kits
DNF-476 & DNF-477

NGS Fragment Kits
DNF-473 & DNF-474

Large Fragment Kits
DNF-464
DNF-492
DNF-493

-X
X

X
-X

Electronic Pipettor
10X at 10 µL volume

Concentration (ng/µL)

Swirl while pipetting up &
down 10X at 2 µL volume

-X
X

1
0.8
0.6
0.4
0.2
1

Well Number

Plate Shaker

1.4
1.2
1
0.8
0.6
0.4
0.2
0

Well Number

Master Mix

1.4
1.2
1
0.8
0.6
0.4
0.2
0

Well Number

Figure 1: A complete row of HS NGS Ladder was run on the Fragment Analyzer™ with the HS NGS Fragment Kit. Bar graph and gel image. (A) No
mixing of the ladder and diluent marker in the plate. (B) Mixing by plate shaker. (C) Master mix of ladder and diluent marker vortexed in a low-bind
tube and then pipetted into each well. Mixing is required for reliable quantification of a sample. The known concentration was 1.025 ng/μL.

Table 1: Recommended mixing protocols for various Fragment Analyzer™ Kits.

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1.2

No Mixing

1.4

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Improved Nucleic Acid QC with Parallel CE

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