Agilent eBook - 17
Best Practices with an Agilent Parallel CE System
5,000
A
4,500
1x TE buffer
4,000
UM
3,500
3,000
2,500
100 mM NaCl
LM
2,000
1,500
1,000
500
Conclusions
Reference
1. Pocernich, C.; Uthe, J.; Pike, W.; Wong, K-S. Best Quantification
Practices with the Agilent 5200 Fragment Analyzer System.
Agilent Technologies Application Note, publication number
5994-0513EN, 2018.
17
| GENengnews.com
6,000
1,000
1,200
1,500
2,000
3,000
700
800
600
500
Size (bp)
B
The 5200 Fragment Analyzer system provides
accurate and consistent sizing for DNA fragments
and smears. Peak size, ideal for fragments, is
automatically provided by ProSize, while the ProSize
Smear Analysis tab provides an average size for
smears based on the distribution of the sample over
the designated smear range. Sizing under 6,000 bp
is accurate and consistent across the concentration
range of the respective kits, with reliable sizing over
6,000 bp occurring with the recommended sample
concentrations. High salt concentrations in samples
minimally affect sizing, still allowing for exceptional
precision and accuracy. n
400
300
100
200
0
1
RFU
The HS NGS DNA Ladder was diluted in 200, 100, 50,
25 mM NaCl, or 1× TE Buffer (10 mM Tris-HCl) and
analyzed on the 5200 Fragment Analyzer system with
the HS NGS Ladder kit (Figure 5). Sizing accuracy
stayed within 3 % error even at the highest salt
concentration. Sizing precision at various salt
concentrations remained excellent throughout the
sizing range of the HS NGS Fragment kit as noted by
the consistently low % CV. Reliable analysis of DNA
samples is ensured with the correct salt concentration.
Sizing of HS NGS DNA Ladder
Known size
(bp)
200 mM NaCl
100 mM NaCl
50 mM NaCl
25 mM NaCl
1× TE Buffer
100
103 ± 0.5
103 ± 0.4
102 ± 0.5
102 ± 0.5
100 ± 0.0
200
204 ± 0.8
204 ± 0.9
203 ± 0.4
203 ± 0.5
201 ± 0.5
300
304 ± 1.2
304 ± 1.3
303 ± 0.5
302 ± 1.0
301 ± 0.4
400
404 ± 1.2
404 ± 1.5
402 ± 0.7
402 ± 1.0
402 ± 0.5
500
504 ± 2.1
504 ± 1.9
503 ± 0.9
502 ± 1.7
502 ± 0.8
600
605 ± 2.4
604 ± 2.4
602 ± 0.8
601 ± 2.1
601 ± 0.7
700
705 ± 4.6
702 ± 2.5
702 ± 1.6
701 ± 1.8
701 ± 1.4
800
806 ± 6.4
804 ± 3.4
803 ± 3.1
802 ± 3.4
802 ± 2.8
900
906 ± 7.2
904 ± 4.0
901 ± 2.3
901 ± 2.4
901 ± 1.9
1,000
1,011 ± 8.8
1,004 ± 4.2
1,001 ± 2.9
1,001 ± 4.1
1,001 ± 2.4
1,200
1,211 ± 15.5
1,205 ± 4.5
1,200 ± 2.4
1,201 ± 6.3
1,200 ± 5.1
1,500
1,512 ± 13.3
1,506 ± 5.6
1,501 ± 0.5
1,499 ± 9.0
1,501 ± 0.5
2,000
2,020 ± 22.5
2,004 ± 7.0
2,001 ± 0.5
1,999 ± 4.2
2,000 ± 0.5
3,000
3,023 ± 19.6
3,023 ± 10.7
2,998 ± 7.2
3,005 ± 10.8
3,001 ± 0.5
Figure 5. HS NGS DNA Ladder analyzed on
the 5200 Fragment Analyzer system with the
HS NGS Fragment kit. (A) Diluted with 100
mM NaCl (red) and the recommended 1× TE
buffer (black). (B) Table displaying average
size (bp) and standard deviation. High salt
concentrations cause a small increase in
sizing. n = 6; LM = lower marker;
UM = upper marker.
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
5994-0585EN
http://www.agilent.com
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