Agilent eBook - 14

Best Practices with an Agilent Parallel CE System

Sizing under 6,000 bp
throughout a dilution series

×103 A
36 ×103
7
6
5
4
3
2
1
0

32
28
24
20

300 bp fragment
350
300

16

Size (bp)

300

RFU

12
8
LM

UM

4

20

0.06 %

-0.3 %

Table 1. Overview of sizing data for 300 and 1,000 bp fragments over
a concentration range separated on the 5200 Fragment Analyzer
system with the HS NGS Fragment kit (1 to 6000 bp). 1 n = 144.

14

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6,000

700
800
1,000
1,500
2,000
3,000

600

500

400

300

200

100

1,200

1,000

Size (bp)

0.33 %

500

800
600
400
200

0

Size (bp)

6,000

0.41 %

UM

700
800
1,000
1,500
2,000
3,000

3.3

LM

600

1.2

1,000

0

500

992 to 1,001

Range (bp)
Standard deviation
% CV
% Error

1

400

297 to 302

Average (bp)

250

1,000 bp fragment

8
4

500

1,200

300

9971

250

3

200

1,000 bp DNF-474

2991

15.6 31.3 62.5 125
Concentration (pg/µL)

4

100

300 bp DNF-474

7.8

×103

1

Sizing over concentration range
4.5 to 600 pg/µL

3.9

Size (bp)

B

900

RFU

12

150

0

2

16

200

50

×103
28
24

250

100

0
1

The HS NGS Fragment kit has a DNA sizing
range of 100 to 6,000 bp. Fragment sizes of
300 and 1,000 bp were analyzed on the 5200
Fragment Analyzer system with the HS NGS
Fragment kit throughout the concentration
range of the kit (Figure 2A & B). The 300 and
1,000 bp fragments reported consistent
sizing of 299 ± 1.24 bp and 997 ± 3.35 bp
respectively, with a % error below 0.3 %. Both
the 300 and the 1,000 bp fragments had
excellent precision values of 0.41 and 0.33 %
CV respectively, indicating consistent sizing
between the concentration points (Table 1).

0

3.9

7.8

15.6 31.3 62.5 125
Concentration (pg/µL)

Figure 2. Sizing of 300 and 1,000 bp fragments over a concentration range on the 5200 Fragment Analyzer system with the HS NGS fragment kit
(1 to 6,000 bp). A) 300 bp fragment overlay (15.6, 62.5, and 500 pg/µL) and bar graph (3.9 to 500 pg/µL)n = 18. B) 1,000 bp fragment overlay (15.6,
62.5, and 500 pg/µL) and bar graph (3.9 to 500 pg/µL)n = 18. Sizing of fragments under 6,000 bp is consistent, and not affected by concentration
on the 5200 Fragment Analyzer system. LM = lower marker; UM = upper marker.


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