Analytical specifications
HS Large Fragment assay
DNA Sizing Range
75 bp – 48,500 bp
DNA Sizing Accuracy1
+ 15% or better
DNA Smear Concentration Range
50 pg/µL -5 ng/µL input DNA (optimal concentration of 1 ng/µL)
DNA Quantification Accuracy2
DNA Quantification Precision2
20% CV
Maximum DNA Concentration
600 pg/µL per fragment; 5 ng/µL per total sample
Physical Specifications3
Total electrophoresis run time
22cm3: 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-464 HS Large
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 HS Large
Fragment 50 kb kit (500 Samples) (Part # DNF-464-0500) is designed for the sizing and quantification of medium to high
molecular weight dsDNA smears/fragments. Example applications include quality control of long read Next Generation
Sequencing (NGS) libraries (e.g., PacBio).
Specifications
1,2
DNA Fragment Concentration Range2
1
Results using DNA Fragment standards at 600 pg/µL and DNA smears at 1 ng/µL prepared from 1x TE buffer.
2
Results using DNA Fragment standards and DNA smears prepared from 1x TE buffer.
3
The 22 cm effective, 47 cm total length capillary is only available for 12-capillary Fragment Analyzer instruments
5 pg/µL – 600 pg/µL input DNA (optimal concentration 500 – 600 pg/µL)
+ 25%
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DNF-464 HS Large Fragment Quick Guide for Fragment Analyzer Systems
1
WARNING
•
•
Kit Components – 500 Sample Kit
Kit Component
Number
Part Number
(Re-order Number)
Description Quantity Per Kit
5191-6568*
DNF-464-FR*
HS Large Fragment 50kb, 500, 4oC
DNF-220-0240 Large Fragment Separation Gel, 240 mL 1
DNF-300-0008 BF-25 Blank Solution, 8mL 1
DNF-355-0125 5x 930 dsDNA Inlet Buffer, 125 mL
• Dilute with sub-micron filtered water prior
to use
DNF-365-U125 HS Extended Large Frag DNA Ladder, 125mL 1
DNF-495-0060 Dilution Buffer 1X TE, 60mL
DNF-497-0125 0.25x TE Rinse Buffer, 125 mL 1
HS LRG Fragment 50kb, FR
DNF-600-U030
DNF-381-0003
Intercalating Dye, 30 μL
HS Large Fragment Diluent Marker, 2.4 mL
1
1
5
5191-6612* Qualitative DNA, RT
C275-130 Eppendorf LoBind 0.5 mL tubes (bag of 50) 1
DNF-475-0050 5x Capillary Conditioning Soln, 50 mL 1
*not orderable
NOTE: 1. The Lambda DNA fragment (48,500 bp) in the HS Extended Large Frag DNA Ladder is sensitive to degradation. The Ladder should be
kept at 2-8°C. Do not freeze or subject to repetitive freeze/thaw cycles. Do not pipette the ladder up and down; vortex with care.
2. The Large Fragment Diluent Marker (DM) solution is provided in aliquots of 2.4 mL vials. To minimize the number of freeze/thaw
cycles, it is highly recommended to work with only one aliquot of DM solution at a time. The DM solution is light and temperature sensitive. For
maximum performance, the DM solution should be kept frozen at –20°C and protected from light when not in use. The DM solution should NOT be
left at room temperature longer than 1 h at a time for sample preparation.
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-464 HS Large 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-464 HS Large 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
running 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 within a day with oil overlay. If
not using right away, cover and keep at 4°C, warm to RT and centrifuge before
4
DNF-464 HS Large 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-464 HS Large Fragment Quick Guide for Fragment Analyzer Systems
WARNING
•
•
5300/5400 system - 96 capillary; Ladder – well H12
Agilent HS Large Fragment DNF-464 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-464 HS Large 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-464-22 – HS Large Fragment 50kb
3.2 DNF-464-33 – HS Large Fragment 50kb
4. Enter Tray Name, Folder Prefix, and Notes (optional).
5. Select OK to add method to the queue.
6. Select to start the separation.
HS Large Fragment Ladder result
High Sensitivity Extended Large Fragment DNA Ladder result, using the Fragment Analyzer system with the DNF-464 High
Sensitivity Large Fragment 50 kb kit. Peaks are annotated by size (bp). Method: DNF-464-33 (33cm “short” array).
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DNF-464 HS Large Fragment Quick Guide for Fragment Analyzer Systems
Issue
Cause
Corrective Action
8,500 bp Lambda DNA fragment in
1 The Ladder was pipetted up and
avoided.
1 Use a new Ladder aliquot and avoid
The peak signal is >> 20,000 RFU;
1 Input DNA sample concentration too
1 Dilute input DNA sample
DNA sample smear overlaps with
1 Input DNA sample size distribution
1 Perform further size selection of
only).
No peak observed for DNA sample
1 Sample concentration too low and
solution or not mixed well.
1 Prepare more concentrated sample
and mixed to sample well.
No sample peak or marker peak
1 Air trapped at the bottom of sample
1 Check sample plate wells for
unclogging a capillary array.
Troubleshooting
The following table lists several potential assay specific issues which may be encountered when using the DNF464 HS Large Fragment 50 kb kit. and suggested remedies. Contact Agilent technical support if you have any
additional troubleshooting or maintenance questions.
the Ladder is degraded or missing.
upper marker peak is low or not
detected relative to lower marker.
Lower/Upper Marker peak.
when expected. Lower/Upper
Marker peaks observed.
down excessively.
2 The Ladder was stored
inappropriately. The Ladder should be
stored at 2-8ºC and freeze-thaw cycles
high. Ensure peak height does not
exceed 2,000 RFU (smear) or 20,000
RFU (fragment), or total input
concentration does not exceed
recommended limits.
outside of assay range.
out of range.
2 Sample not added to Diluent Marker
pipetting the Ladder up and down
excessively.
2 Store and handle the Ladder as
directed in this User Manual.
concentration with supplied Dilution
Buffer 1x TE (DNF-495) and repeat
experiment; OR Repeat experiment
using decreased injection time or
voltage.
sample to narrow DNA size
distribution and repeat experiment;
OR repeat experiment using DNF488 HS gDNA kit (uses lower marker
and repeat experiment: OR repeat
experiment using increased injection
time and/or injection voltage.
2 Verify sample was correctly added
observed for individual sample.
plate well, or bubbles present in
sample well.
2 Insufficient sample volume. A
minimum of 20 µL is required.
3 Capillary is plugged.
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
Appendix – Capillary Array Cleaning of
the Fragment Analyzer User Manual for
8
DNF-464 HS Large Fragment Quick Guide for Fragment Analyzer Systems
Marker and/or Ladder peaks are
longer than expected,
1 Capillary array needs to be
1 Flush array with 0.5 N NaOH
water. (See User Manual for details).
48,500bp Lambda DNA fragment
1 Occasional Ladder lot variations
1 Manually delete extra peak migrating
NOTE:
assigned in the Ladder.
broad, signals are lower than
expected, and/or migration time
peak is the Ladder is split and/or
not assigned properly in the
software.
reconditioned.
2 Capillary array vent valve is clogged.
may result in secondary peak
appearing before main Lambda DNA
peak.
solution and repeat experiment.
2 Clean vent valve with deionized
before main Lambda DNA peak or
increase peak height threshold in
ladder well to not call extra peak.
Sample sizing and
quantification will not be affected by
the presence of the extra peak, if the
Lambda DNA peak is correctly
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 10/20
SD-AT000127
9
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