Agilent eBook - 7
Best Practices with an Agilent Parallel CE System
7
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3,000
2,000
1,500
1.0
1,000
900
800
700
Size (bp)
1.2
0.8
0.6
0.4
1.4
600
500
400
300
200
100
0.2
1
1
2
3
4
5
6
7
8
Well number
9
10
11
12
Plate shaker
A1
A2
A3
A4
A5
A6
A7
Well number
A8
A9
A10
A11
A12
A1
A2
A3
A4
A5
A6
A7
Well number
A8
A9
A10
A11
A12
A1
A2
A3
A4
A5
A6
A7
Well number
A8
A9
A10
A11
A12
6,000
3,000
2,000
1,500
1,000
900
800
700
1.2
1.0
600
0.8
0.6
0.4
1.4
500
400
300
200
0.2
0
C
6,000
Size (bp)
Concentration (ng/µL)
B
No mixing
100
1
1
2
3
4
5
6
7
Well number
8
9
10
11
Master mix
6,000
3,000
2,000
1,500
1,000
900
800
700
1.2
1.0
Size (bp)
The Agilent HS NGS DNA Ladder was utilized as the
sample for the comparison of the mixing techniques.
The concentration was determined to be 1.025 ng/µL
on the Qubit 2.0 with the dsDNA HS kit. The HS NGS
Diluent Marker (1-6000 bp), then sample were individually
pipetted into each well of an entire row on a 96-well
plate with no additional mixing, followed by separation
on the Agilent 5200 Fragment Analyzer system with
the Agilent HS NGS Fragment kit. After the initial 'no
mixing' analysis, the same plate was then vortexed at
3,000 rpm for 2 minutes, followed by analysis on the
Agilent 5200 Fragment Analyzer system. Data from
samples not mixed showed various concentrations
ranging from 0.17 to 0.81 ng/µL resulting in a high
standard deviation, low accuracy, and extremely poor
precision (high % CV) (Table 2). Mixing the sample
plate with the plate shaker improved the homogeneity
1.4
0
Concentration (ng/µL)
Standard deviation, precision, and accuracy were
the parameters for comparing the mixing methods.
Standard deviation is used to quantify the amount of
variation or dispersion of a set of data values from the
mean. Accuracy measures the closeness of a number
to the true value and is reported as % error. Precision
is the closeness of two or more measurements and
is independent of accuracy. A low % CV, representing
precision, demonstrates the closeness of the
measurements and reliability of the average.
A
Concentration (ng/µL)
demonstrate the effects mixing has on quantification
(Figure 1).
0.8
0.6
600
500
400
300
0.4
200
0.2
100
0
1
1
2
3
4
5
6
7
Well number
8
9
10
11
Figure 1. A complete row of Agilent HS NGS DNA Ladder was run on the Agilent 5200 Fragment Analyzer system with the Agilent HS NGS Fragment kit
(1‑6000 bp). 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.
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Agilent eBook
Table of Contents for the Digital Edition of Agilent eBook
Agilent eBook - 1
Agilent eBook - 2
Agilent eBook - 3
Agilent eBook - 4
Agilent eBook - 5
Agilent eBook - 6
Agilent eBook - 7
Agilent eBook - 8
Agilent eBook - 9
Agilent eBook - 10
Agilent eBook - 11
Agilent eBook - 12
Agilent eBook - 13
Agilent eBook - 14
Agilent eBook - 15
Agilent eBook - 16
Agilent eBook - 17
Agilent eBook - 18
Agilent eBook - 19
Agilent eBook - 20
Agilent eBook - 21
Agilent eBook - 22
Agilent eBook - 23
Agilent eBook - 24
Agilent eBook - 25
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