Improved Nucleic Acid QC with Parallel CE - 7

Application Note

Application Note

Best Sizing Practices

Best Sizing Practices

Effects of Salt

300 bp Fragment

Size (bp)

Awareness of the salt concentration in samples is important for consistent results. The Fragment Analyzer sample preparation protocol
recommends that all samples are diluted with 1X 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.

LM

The HS NGS ladder was diluted in 200, 100, 50, 25 mM NaCl or 1X TE Buffer (10 mM Tris-HCl) and analyzed on the Fragment Analyzer
with the HS NGS Fragment 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.

UM

Concentration pg/µL

A

1,000 bp Fragment

Size (bp)

1X TE Buffer

LM

LM
100 mM NaCl

UM

Concentration pg/µL
Figure 2: Sizing of 300 bp and 1,000 bp fragments over a concentration range on the Fragment Analyzer™ with the HS NGS Fragment Kit. (A) 300 bp
fragment overlay (15.6, 62.5, & 500 pg/µL) and bar graph (3.9 - 500 pg/µL). n=18. (B) 1,000 bp fragment overlay (15.6, 62.5, & 500 pg/µL) and bar graph
(3.9 - 500 pg/µL). n=18. Sizing of fragments under 6,000 bp is consistent and not affected by concentration on the Fragment Analyzer. LM = Lower
Marker, UM = Upper Marker.

A

UM

Size (bp)

B

B

Sizing of HS NGS Ladder
Known Size
(bp)

200 mM NaCl

100 mM NaCl

50 mM NaCl

25 mM NaCl

1X 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

3.2%

500

504 ± 2.1

504 ± 1.9

503 ± 0.9

502 ± 1.7

502 ± 0.8

Figure 3: DNA smear separated by the Fragment Analyzer™ with the HS Small Fragment Kit over a concentration range of 5,300 - 78 pg/µL.
(A) Electropherogram overlay. (B) Table of average smear size, standard deviation, and precision (%CV). DNA smear sizing remained consistent
over the entire concentration range. n=10, LM = Lower Marker, UM = Upper Marker.

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

Smear Size Over Concentration Range

UM

Concentration
5,300 2,900 1,350
(pg/µL)

LM

A

320

160

78

Average (bp)

295

294

294

294

296

296

295

Std. Dev.

3.5

2.6

3.2

4.3

6.2

5.9

9.4

Precision
%CV

1.2%

0.9%

1.1%

B

1.5% 2.1% 2.0%

DNF-464 HS Large Fragment Kit
15,000 bp
at 600 pg/μL

14,566

Average (bp)
LM

650

UM

Range (bp)

14,566
14,417 - 14,804

Std. Dev.

208.0

%CV

1.4%

%Error

-2.9%

Figure 5: HS NGS ladder analyzed on the Fragment Analyzer™ with the HS NGS Fragment Kit. (A) Diluted with 100 mM NaCl (red) and the
recommended 1X 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.

Figure 4: Sizing of 15,000 bp fragment at 600 pg/µL on the Fragment Analyzer™ with the HS Large Fragment Kit. (A) Electropherogram. (B) Table of
15,000 bp fragment sizing data. n=3, LM = Lower Marker, UM = Upper Marker.

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