Analytical specifications
HS NGS Fragment assay
Sizing Range
100 bp – 6,000 bp
Sizing Accuracy2
+ 5% or better
Sizing Precision2
2% CV
Fragment Concentration Range2
5 pg/µL – 500 pg/µL input DNA
Smear Concentration Range
50 pg/µL – 5,000 pg/µL input DNA
Quantification Accuracy
+ 25%
Quantification Precision
15% CV
Quantitative Range
Fragment3:5 - 500 pg/µL; Smear3:50 – 5,000 pg/µL
Physical Specifications
Total electrophoresis run time
22cm1: 25 minutes, 33cm: 50 minutes, 55cm: 80 minutes
Samples per run
12, 48 or 96; depending on the instrument type
Sample volume required
2 µL
Kit stability
4 months
Agilent DNF-474 HS NGS
Fragment Kit Quick Guide for
Fragment Analyzer Systems
The Agilent Fragment Analyzer systems are automated capillary electrophoresis platforms for scalable,
flexible, fast, and reliable electrophoresis of nucleic acids.
This Quick Guide is intended for use with the Agilent 5200, 5300, and 5400 Fragment Analyzer systems only.
The DNF-474 HS NGS Fragment assay is designed for the quantitative and qualitative analysis of NGS libraries
and their intermediates from 100 to 6,000 bp.
Specifications
1,2
Separation Resolution
2,3
2,3
1
The FA 12-Capillary Array Ultrashort, 22 cm is only available for 5200 Fragment Analyzer system.
2
Results using DNA ladder in 1X TE buffer.
3
Results using DNA samples in 1X TE buffer.
100 bp – 2,000 bp < 5%
2,000 bp – 5,000 bp < 10%
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DNF-474 HS NGS Fragment Quick Guide for Fragment Analyzer Systems
•
•
Kit Components – 500 Sample Kit
Kit Component
Number
Part Number
(Re-order Number)
Description Quantity Per Kit
5191-6578*
DNF-240-0240 NGS Separation Gel, 240 mL 1
DNF-300-0008 BF-25 Blank Solution, 8mL 1
DNF-355-0125 5x 930 dsDNA Inlet Buffer, 125 mL
DNF-497-0125 0.25x TE Rinse Buffer, 125 mL 1
DNF-474-FR*
DNF-600-U030
DNF-373-0003
DNF-396-U100 HS NGS DNA Ladder, 100 μL 1
DNF-475-0050 DNF-475-0050 5x Capillary Conditioning Soln, RT
*Not orderable.
HS NGS Fragment (1-6000bp), 500, 4oC
• Dilute with sub-micron filtered water prior
to use
HS NGS Frag (1-6000bp), -20oC
Intercalating Dye, 30 μL
HS NGS Diluent Marker (1-6000bp), 2.4 mL
• Dilute with sub-micron filtered water prior
to use
1
1
5
1
WARNING
Refer to product safety data sheets for further information
When working with the Fragment Analyzer kit components follow the appropriate safety
procedures such as wearing goggles, safety gloves and protective clothing.
2
DNF-474 HS NGS Fragment Quick Guide for Fragment Analyzer Systems
•
•
Kit Components – 1000 Sample Kit
Kit Component
Number
Part Number
(Re-order Number)
Description Quantity Per Kit
5191-5679*
DNF-240-0500 NGS Separation Gel, 500 mL 1
DNF-300-0008 BF-25 Blank Solution, 8mL 1
DNF-355-0300 5x 930 dsDNA Inlet Buffer, 300 mL
DNF-497-0125 0.25x TE Rinse Buffer, 125 mL 1
DNF-476-FR*
DNF-600-U030
DNF-362-0003
DNF-363-U100 HS NGS DNA Ladder, 100 μL 2
DNF-475-0100 DNF-475-0100 5x Capillary Conditioning Soln, RT
*Not orderable.
HS NGS Fragment (1-6000bp), 1000, 4oC
• Dilute with sub-micron filtered water prior
to use
HS NGS Frag (1-6000bp), -20oC
Intercalating Dye, 30 μL
HS NGS Diluent Marker (1-6000bp), 2.4 mL
• Lower marker (set to 1 bp) and 6,000 bp
upper marker
• Fragments from 100 bp – 3,000 bp;
1 ng/µL total DNA concentration
• Dilute with sub-micron filtered water prior
to use
1
2
10
1
WARNING
Refer to product safety data sheets for further information
When working with the Fragment Analyzer kit components follow the appropriate safety
procedures such as wearing goggles, safety gloves and protective clothing.
3
DNF-474 HS NGS Fragment Quick Guide for Fragment Analyzer Systems
Additional Material Required for Analysis with the Fragment Analyzer Systems
• Fragment Analyzer systems with LED fluorescence detection:
• 5200 Fragment Analyzer system (p/n M5310AA)
• FA 12-Capillary Array Ultrashort, 22 cm (p/n A2300-1250-2247) OR
• FA 12-Capillary Array Short, 33 cm (p/n A2300-1250-3355) OR
• FA 12-Capillary Array Long, 55 cm (p/n A2300-1250-5580)
• 5300 Fragment Analyzer system (p/n M5311AA)
• FA 48-Capillary Array Short, 33 cm (p/n A2300-4850-3355) OR
• FA/ZAG 96-Capillary Array Short, 33 cm (p/n A2300-9650-3355) OR
• FA/ZAG 96-Capillary Array Long, 55 cm (p/n A2300-9650-5580)
• 5400 Fragment Analyzer system (p/n M5312AA)
• FA 48-Capillary Array Short, 33 cm (p/n A2300-4850-3355) OR
• FA/ZAG 96-Capillary Array Short, 33 cm (p/n A2300-9650-3355) OR
• FA/ZAG 96-Capillary Array Long, 55 cm (p/n A2300-9650-5580):
• Agilent Fragment Analyzer controller software (Version 1.1.0.11 or higher)
• Agilent ProSize data analysis software (Version 2.0.0.61 or higher)
Additional equipment/reagents required (not supplied)
• 96-well PCR sample plates. Please refer to Appendix – Fragment Analyzer Compatible Plates and Tubes in the Fragment
Analyzer System User Manual for a complete approved sample plate list
• Multichannel pipettor(s) and/or liquid handling device capable of dispensing 1 – 100 µL volumes (sample plates) and
1,000 µL volumes (inlet buffer plate)
• Pipette tips
• 96-well plate centrifuge (for spinning down bubbles from sample plates)
• Sub-micron filtered DI water system (for diluting the 5x 930 dsDNA Inlet Buffer and 5x Capillary Conditioning Solution)
• 96-deepwell 1mL plate: Fisher Scientific #12-566-120 (inlet buffer and/or waste plate)
• Reagent reservoir, 50 mL (VWR #89094-680 or similar) (for use in pipetting inlet buffer plates/sample trays)
• Conical centrifuge tubes for prepared separation gel/dye mixture and/or 1x Capillary Conditioning Solution
• 50 mL (for 5200 Fragment Analyzer system or 50 mL volumes): BD Falcon #352070, available from Fisher
Scientific #14-432-22 or VWR #21008-940
• 250 mL (for 5300 and 5400 Fragment Analyzer systems or larger volumes): Corning #430776, available from Fisher
Scientific #05-538-53 or VWR #21008-771
• Vortexer (for mixing of samples, ladders, and/or markers in tubes and/or plates)
Capillary Storage Solution (p/n GP-440-0100)
•
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DNF-474 HS NGS Fragment Quick Guide for Fragment Analyzer Systems
Keep reagents during sample preparation at room temperature
Ensure that no sample or Diluent Marker remains within or on the outside of the tip
• Apply a new seal to 96-well sample plate prior to mixing and centrifugation
plate
Essential Measurement Practices
Environmental
conditions
Steps before
sample preparation
Pipetting practice
• Ambient operating temperature: 19 – 25 °C (66 – 77 °F)
•
• Allow reagents to equilibrate at room temperature for 30 min prior to use
• Pipette reagents carefully against the side of the 96-well sample plate or sample
tube
•
• When mixing sample with Diluent Marker (DM), it is important to mix the contents of
the well thoroughly to achieve the most accurate quantification. It is highly
suggested to perform one of the following methods to ensure complete mixing.
After mixing, briefly centrifuge and visually confirm that all liquid is collected at the
bottom of the 96-well sample plate or tube strips and any air bubble is removed
• After adding 2 µL of sample or ladder to the 22 µL of DM, place a plate seal on
the sample plate and vortex the sample plate at 3,000 rpm for 2 min. Any
suitable benchtop plate vortexer can be used. Ensure that there is no well-towell transfer of samples when vortexing. The plate should be spun via a
Mixing and
centrifugation
recommendations
centrifuge after vortexing to ensure there are no trapped air bubbles in the
wells.
• After adding 2 µL of sample or ladder to the 22 µL of DM, use a separate
pipette tip set to a larger 20 µL volume, and pipette each well up/down to
further mix.
• Use an electronic pipettor capable of mixing a 10 µL volume in the tip after
dispensing the 2 µL sample or ladder volume. Some models enable using the
pipette tip for both adding and mixing.
• Run samples immediately after preparation, or within a day with oil overlay. If not
using right away, cover and keep at 4°C, warm to RT and centrifuge before running
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DNF-474 HS NGS Fragment Quick Guide for Fragment Analyzer Systems
12
1.0 µL
10 mL
10 mL
24
1.5 µL
15 mL
15 mL
36
2.0 µL
20 mL
20 mL
48
2.5 µL
25 mL
25 mL
96
4.5 µL
45 mL
45 mL
48
2.5 µL
25 mL
25 mL
96
4.0 µL
40 mL
40 mL
144
5.5 µL
55 mL
55 mL
192
7.0 µL
70 mL
70 mL
240
8.5 µL
85 mL
85 mL
288
10.0 µL
100 mL
100 mL
96
4.0 µL
40 mL
40 mL
192
8.0 µL
80 mL
80 mL
288
12.0 µL
120 mL
120 mL
384
16.0 µL
160 mL
160 mL
480
20.0 µL
200 mL
200 mL
Gel preparation
Prepare gel/dye mixture for 5200, 5300, and 5400 Fragment Analyzer Systems. To ensure the gel/dye mixture is mixed homogeneously without generating bubbles, gently invert the centrifuge tube 5 to 10 times, depending on the volume of the mixture.
5200 Fragment Analyzer system volume specifications
# of Samples to be
Analyzed
1
1
One sample well per separation is dedicated to the ladder.
2
A 5 mL minimum volume in the tube is included.
Volume of Intercalating
Dye
Volume of Separation
2
Gel
Volume of 1x Conditioning
Solution
2
5300 Fragment Analyzer system volume specifications with 48-capillary array
# of Samples to be
Analyzed
1
1
One sample well per separation is dedicated to the ladder.
2
A 5 mL minimum volume in the tube is included.
Volume of Intercalating
Dye
Volume of Separation
2
Gel
Volume of 1x Conditioning
2
Solution
5300 and 5400 Fragment Analyzer systems volume specifications with 96-capillary arrays
# of Samples to be
Analyzed
1
1
One sample well per separation is dedicated to the ladder.
2
A 5 mL minimum volume in the tube is included.
Volume of Intercalating
Dye
Volume of Separation
2
Gel
Volume of 1x Conditioning
Solution
2
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DNF-474 HS NGS Fragment Quick Guide for Fragment Analyzer Systems
WARNING
•
•
5300/5400 system - 96 capillary; Ladder – well H12
Agilent HS NGS Fragment DNF-474 assay operating procedure
1. Mix fresh gel and dye according to the volumes in the Gel preparation tables. Refill 1x Capillary Conditioning
Solution as needed.
2. Place a fresh 1x 930 dsDNA Inlet Buffer in drawer ‘B’ on the system, 1.0 mL/well. Replace daily.
2.1. 5200 system; Fill row A of buffer plate
2.2. 5300 system - 48 capillary; Fill rows A-D of buffer plate
2.3. 5300/5400 system - 96 capillary; Fill all rows of buffer plate
3. Prepare Capillary Storage Solution plate. Replace every 2-4 weeks for optimal results.
3.1. 5200 system; Fill row H of buffer plate with 1.0mL/well, place in drawer “B “
3.2. 5300 system - 48 capillary; Fill rows A-D of a sample plate with 100 µL/well, place in drawer ‘3’
3.3. 5300/5400 system - 96 capillary; Fill all rows of a sample plate with 100 µL/well, place in drawer ‘3’
4. Place 0.25x TE Rinse Buffer plate in drawer ‘M’ on the system, 200 µL/well. Replace daily.
4.1. 5200 system; Fill row A of sample plate
4.2. 5300 system - 48 capillary; Fill rows A-D of sample plate
4.3. 5300/5400 system - 96 capillary; Fill all rows of sample plate
5. Mix samples or Ladder with Diluent Marker in sample plate, add 24 µL of BF-25 Blank Solution to
unused wells. Place ladder in corresponding well dependent on the capillary size.
3.3.1. 5400 system; place in drawer “S”
Working with Chemicals
The handling of reagents and chemicals might hold health risks.
Refer to product material safety datasheets for further chemical and biological safety information.
Follow the appropriate safety procedures such as wearing goggles, safety gloves and protective clothing.
5200 system; Ladder – well 12, depending on which
row is chosen
5300 system - 48 capillary; Ladder – well D12 or H12,
depending on which group is chosen
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DNF-474 HS NGS Fragment Quick Guide for Fragment Analyzer Systems
Agilent Fragment Analyzer software operating procedure
1. Select Row, Group or Tray to run.
2. Enter sample ID and Tray ID (optional).
3. Select Add to Queue, from the dropdown menus select the corresponding method based on your capillary length;
3.1 DNF-474-22 – HS NGS Fragment 1-6000bp
3.2 DNF-474-33 – HS NGS Fragment 1-6000bp
3.3 DNF-474-55 – HS NGS Fragment 1-6000bp
4. Enter Tray Name, Folder Prefix, and Notes (optional).
5. Select OK to add method to the queue.
6. Select to start the separation.
DNA Ladder result
Representative NGS DNA Ladder result using the Fragment Analyzer system with the DNF-474 HS NGS Fragment kit (1bp –
6,000bp). Method: DNF-474-33 (short array). Peaks annotated by size (bp).
8
DNF-474 HS NGS Fragment Quick Guide for Fragment Analyzer Systems
Issue
Cause
Corrective Action
The peak signal is >> 20,000 RFU;
1 Input DNA sample concentration is
1 Dilute input DNA sample
range from 5 – 100 ng/µL.
DNA Sample smear overlaps with
1 Input DNA sample size distribution
1 Perform further size selection of
and mixed in sample well.
No sample peak or marker peak
unclogging a capillary array.
Troubleshooting
The following table lists several potential assay specific issues which may be encountered when using the HS NGS
Fragment kit (1-6000 bp) (Part #DNF-474) and suggested remedies. Contact Agilent technical support if you have any
additional troubleshooting or maintenance questions.
upper marker peak is low or not
detected relative to lower marker.
Lower/Upper Marker peak.
observed for individual sample.
too high. Ensure total signal height
does not exceed 20,000 RFU, or total
input DNA concentration does not
far exceed 5,000 pg/µL.
outside of assay range.
2 Input DNA sample concentration too
high.
1 Air trapped at the bottom of the
sample plate well, or bubbles present
in sample well.
2
Insufficient sample volume. A
minimum of 20µL is required.
3
Capillary is plugged.
concentration with 1x TE buffer and
repeat experiment; OR
Repeat experiment using decreased
injection time (e.g., 10 sec); OR
Prepare fresh sample using NGS
Fragment kit (1-6000 bp) (Part #
DNF-473), which covers input DNA
sample to narrow DNA size
distribution and repeat experiment;
OR
Prepare fresh sample using HS
Large Fragment Kit (part #DNF-493)
2 Verify sample was correctly added
1 Check sample plate wells for
trapped air bubbles. Centrifuge
plate.
2
Verify proper volume of solution
was added to sample well
3 Check waste plate for liquid in the
capillary well. If no liquid is
observed, follow the steps outlined
in the System Manual for
Technical Support and Further Information
For technical support, please visit www.agilent.com. It offers useful information, support and current developments about the
products and technology.
www.agilent.com
© Agilent Technologies, Inc. 2020
Edition 01/21
SD-AT000134
9
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