Agilent eBook - 16

Best Practices with an Agilent Parallel CE System

Sizing over 6,000 bp

Effects of salt

Awareness of the salt concentration in samples
is important for consistent results. The 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 that the chloride salt concentration
in the sample remains less than 10 mM. High
salt concentrations may cause noisy baselines,
sporadic spikes in the electropherogram, and
decreased quantification1.

16

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A

8,000
7,000
RFU

14,566

6,000
5,000
4,000
3,000

UM

LM

2,000

B

200,000

48,500

15,000

6,000

10,000

Size (bp)

3,000

1,500

1,000

800

600

400

200

0

75

1,000
1

The HS Large Fragment 50 kb kit has a
DNA sizing range of 75 to 48,500 bp. To achieve
the most reliable and accurate sizing with HS
Large Fragment 50 kb kit, an optimal fragment
concentration range of 500 to 600 pg/µL and
an optimal smear concentration of 1 ng/µL is
recommended. A 15,000 bp fragment was
separated on the 5200 Fragment Analyzer
system with the HS Large Fragment 50 kb kit at
the recommended concentration of 600 pg/µL
(Figure 4). An average size of 14,566 bp was
reported with high accuracy (2.9 % error) and
precision (1.4 % CV).

9,000

HS Large Fragment 50kb kit
15,000 bp at 600 pg/μL
Average (bp)
Range (bp)

14,566
14,417 to 14,804

Standard deviation

208.0

% CV

1.4 %

% Error

-2.9 %

Figure 4. Sizing of 15,000 bp fragment at 600 pg/µL on the
5200 Fragment Analyzer system with the HS Large Fragment 50 kb kit.
(A) Electropherogram. (B) Table of 15,000 bp fragment sizing data. n = 3;
LM = lower marker; UM = upper marker.



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Agilent eBook

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