Analytical specifications
dsDNA 915 assay
DNA Sizing Range
35 bp – 5,000 bp
DNA Sizing Accuracy1
+ 5% or better
DNA Fragment Concentration Range1
DNA Sizing Precision1
2% CV
Physical Specifications
Total electrophoresis run time
22 cm2: 22 minutes, 33 cm: 45 minutes, 55 cm: 70 minutes
Samples per run
12, 48 or 96; depending on the instrument type
Sample volume required
2 µL (adjustable depending on sample concentration)
Kit stability
4 months
Agilent DNF-915 Reagent Kit
(35 – 5000 bp) 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 dsDNA 915
Reagent kit from Agilent is for the analysis of dsDNA fragments between 35 and 5,000 bp. Sizing and relative quantification
between samples can be obtained using this kit. Example applications include general PCR fragment sizing and QC, and
genotyping.
Specifications
0.5 ng/µL – 50 ng/µL input DNA (adjustable by dilution sample)
Separation Resolution
1
Results using DNA Ladder of DNA Fragment standards initially prepared in 1x TE buffer.
2
The 22 cm effective, 47 cm total length capillary is only available for 12-capillary Fragment Analyzer instruments
35 –100 bp ≤ 10%; 100 –700 bp ≤ 5%; 700 -5,000 bp ≤ 10% (22-47 Array)
35 –100 bp ≤ 10%; 100 – 1,000 bp ≤ 5%; 1,000 – 5,000 bp ≤10% (33-55 Array)
35 –1,200 bp ≤ 5%; 1,200 –5,000 bp ≤ 6% (55-80 Array)
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DNF-915 dsDNA Reagent Kit (35-5000 bp) Quick Guide for Fragment Analyzer
1
WARNING
•
•
Kit Components – 500 Sample Kit
Kit Component
Number
Part Number
(Re-order Number)
Description Quantity Per Kit
5191-6595*
DNF-810-0240 dsDNA 810 Gel, 240 mL 1
DNF-355-0125 5x 930 dsDNA Inlet Buffer, 125 mL
DNF-495-0060 Dilution Buffer 1X TE, 60mL
DNF-915-FR*
DNF-600-U030
FS-SLR915-0001
FS-SMK480-0003
5191-6614* Qualitative DNA, 500, RT
FS-SMO15 Mineral Oil Dropper Bottle, 15mL 1
DNF-475-0050 5x Capillary Conditioning Soln, 50 mL 1
dsDNA 910/915, 500, 4oC
• Dilute with sub-micron filtered water prior
to use
dsDNA 915 (35-5000), FR
Intercalating Dye, 30 μL
100bp DNA Plus Ladder, 1mL
Markers, 35bp & 5000bp, 3.2 mL
1
1
1
1
*not orderable
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-915 dsDNA Reagent Kit (35-5000 bp) Quick Guide for Fragment Analyzer
1
WARNING
•
•
Kit Components – 1000 Sample Kit
Kit Component
Number
Part Number
(Re-order Number)
Description Quantity Per Kit
5191-6596*
DNF-810-0500 dsDNA 910 Gel, 500 mL 1
DNF-355-0300 5x 930 dsDNA Inlet Buffer, 300 mL
DNF-495-0125 Dilution Buffer 1X TE, 125mL
DNF-915-FR*
DNF-600-U030
FS-SLR915-0001
FS-SMK480-0003
5191-6615* Qualitative DNA, 1000-5000, RT
FS-SMO15 Mineral Oil Dropper Bottle, 15mL 1
DNF-475-0100 5x Capillary Conditioning Soln, 100 mL 1
*not orderable
dsDNA 910/915, 1000, 4oC
• Dilute with sub-micron filtered water prior
to use
dsDNA 915 (35bp-5000bp), FR
Intercalating Dye, 30 μL
100bp DNA Plus Ladder, 1mL
Markers, 35bp & 5000bp, 3.2 mL
1
2
2
2
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-915 dsDNA Reagent Kit (35-5000 bp) Quick Guide for Fragment Analyzer
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)
•
4
DNF-915 dsDNA Reagent Kit (35-5000 bp) Quick Guide for Fragment Analyzer
Ambient operating temperature: 19 – 25 °C (66 – 77 °F)
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
•
•
• 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-915 dsDNA Reagent Kit (35-5000 bp) Quick Guide for Fragment Analyzer
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
6
DNF-915 dsDNA Reagent Kit (35-5000 bp) Quick Guide for Fragment Analyzer
WARNING
•
•
Follow the appropriate safety procedures such as wearing goggles, safety gloves and protective clothing.
5300/5400 system - 96 capillary; Ladder – well H12
Agilent dsDNA 915 (35-5000bp) DNF-915 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. Prepare Marker plate and place in drawer ‘M’ on the system, 30 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 with Diluent Buffer 1x TE in sample plate. Add ready to use ladder in corresponding well,
dependent on the capillary size .
3.3.1. 5400 system; place in drawer “S”
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
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.
7
DNF-915 dsDNA Reagent Kit (35-5000 bp) Quick Guide for Fragment Analyzer
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-915-22 – DNA 35-5000 bp
3.2 DNF-915-33 – DNA 35-5000 bp
3.3 DNF-915-55 – DNA 35-5000 bp
4. Enter Tray Name, Folder Prefix, and Notes (optional).
5. Select OK to add method to the queue.
6. Select to start the separation.
100 bp Plus DNA Ladder result
Representative 100 bp Plus DNA Ladder result co-injected with 35 bp lower marker and 5,000 bp upper marker, using the
Fragment Analyzer system with the dsDNA 915 Reagent kit. Method: DNF-915-22 (“ ultrashort” array).
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DNF-915 dsDNA Reagent Kit (35-5000 bp) Quick Guide for Fragment Analyzer
Issue
Cause
Corrective Action
The peak signal is >> 20,000 RFU;
1
Input DNA sample concentration is
1
Further dilute input DNA sample
quantification accuracy.
Sample peak(s) migrate before or
1
Excess primer-dimer species in
1
Further dilute input DNA sample
and repeat experiment.
Sample peak(s) migrate after or co-
1 DNA sample size out of range of
1 Analyze samples with dsDNA 920
20,000 bp (DNF-930)
No peak observed for DNA sample
1 Sample concentration too low and out
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
array.
Troubleshooting
The following table lists several potential assay specific issues which may be encountered when using the dsDNA 915 Reagent
kit 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.
co-migrate with 35 bp Lower Marker.
migrate with 5,000 bp Upper Marker.
when expected. Lower/Upper Marker
peaks observed.
too high.
assay.
of range.
2 Sample was not added to 1x TE
diluent or not mixed well.
sample.
concentration with 1x TE buffer and
repeat experiment.
2
Reduce injection time and/or injection
voltage and repeat experiment. Use
the same injection voltage/time
settings for the Marker Plate and
Sample Plate to maximize
concentration with 1x TE buffer to
minimize primer-dimer interference
Reagent kit, 75 – 15,000 bp (DNF-
920), or dsDNA 930 Reagent kit, 75 –
and repeat experiment (e.g. 4 μL
sample + 20 μL DI water); OR Repeat
experiment using increased injection
time and/or injection voltage for
Marker Plate and Sample Plate.
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 unclogging a capillary
9
DNF-915 dsDNA Reagent Kit (35-5000 bp) Quick Guide for Fragment Analyzer
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-AT000122
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