Agilent DNF-935 Quick Start Guide

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Analytical specifications

dsDNA 935 assay

DNA Sizing Range

100 bp – 1,500 bp

DNA Sizing Accuracy1

+ 15% or better

DNA Fragment Concentration Range1

DNA Sizing Precision1

2% CV

Physical Specifications

Total electrophoresis run time

22cm2: 15 minutes, 33cm: 20 minutes, 55cm: 35 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-935 Reagent Kit (1 – 1500 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 DNF-935 Reagent kit from Agilent is for the fast analysis of dsDNA fragments between 100 bp and 1,500 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

Results using DNA Ladder of DNA Fragment standards initially prepared in 1x TE buffer.

The 22 cm effective, 47 cm total length capillary is only available for 12-capillary Fragment Analyzer instruments

1 bp – 100 bp ≤ 15%; 100 bp – 1,500 bp ≤ 10% (22-47 Array) 1 bp – 100 bp ≤ 15%; 100 bp – 1,500 bp ≤ 10% (33-55 Array) 1 bp – 1,500 bp ≤ 5% (55-80 Array)

DNF-935 dsDNA Reagent Kit (1-1500 bp) Quick Guide for Fragment Analyzer

WARNING

•

Kit Components – 500 Sample Kit

Kit Component Number

Part Number (Re-order Number)

Description Quantity Per Kit

5191-6599*

DNF-930-0240 dsDNA 930 Gel, 240 mL 1

DNF-355-0125 5x 930 dsDNA Inlet Buffer, 125 mL

DNF-495-0060 Dilution Buffer 1X TE, 60mL

DNF-935-FR*

DNF-600-U030

FS-SLR910-0001

FA-MRK910F-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 930/935, 500, 4oC

• Dilute with sub-micron filtered water prior to use

dsDNA 935, FR

Intercalating Dye, 30 μL

100bp DNA Ladder, 100 µL

Markers, 1bp & 1500kb, 3.2 mL

*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.

DNF-935 dsDNA Reagent Kit (1-1500 bp) Quick Guide for Fragment Analyzer

WARNING

•

Kit Components – 1000 Sample Kit

Kit Component Number

5191-6600*

DNF-930-FR*

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

Part Number (Re-order Number)

DNF-930-0500 dsDNA 930 Gel, 500 mL 1

DNF-355-0300 5x 930 dsDNA Inlet Buffer, 300 mL

DNF-495-0125 Dilution Buffer 1X TE, 125 mL

DNF-600-U030

FS-SLR910-0001

FA-MRK910F-0003

Description Quantity Per Kit

dsDNA 930/935, 1000, 4oC

• Dilute with sub-micron filtered water prior to use

dsDNA 930, FR

Intercalating Dye, 30 μL

100bp DNA Ladder, 100 µL

Markers, 1bp & 1500kb, 3.2 mL

*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.

DNF-935 dsDNA Reagent Kit (1-1500 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)

•

DNF-935 dsDNA Reagent Kit (1-1500 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

•

• Allowreagents 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

DNF-935 dsDNA Reagent Kit (1-1500 bp) Quick Guide for Fragment Analyzer

1.0 µL

10 mL

1.5 µL

15 mL

2.0 µL

20 mL

2.5 µL

25 mL

4.5 µL

45 mL

2.5 µL

25 mL

4.0 µL

40 mL

144

5.5 µL

55 mL

192

7.0 µL

70 mL

240

8.5 µL

85 mL

288

10.0 µL

100 mL

4.0 µL

40 mL

192

8.0 µL

80 mL

288

12.0 µL

120 mL

384

16.0 µL

160 mL

480

20.0 µL

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

One sample well per separation is dedicated to the ladder.

A 5 mL minimum volume in the tube is included.

Volume of Intercalating Dye

Volume of Separation

Gel

Volume of 1x Conditioning Solution

5300 Fragment Analyzer system volume specifications with 48-capillary array

# of Samples to be Analyzed

One sample well per separation is dedicated to the ladder.

A 5 mL minimum volume in the tube is included.

Volume of Intercalating Dye

Volume of Separation

Gel

Volume of 1x Conditioning

Solution

5300 and 5400 Fragment Analyzer systems volume specifications with 96-capillary arrays

# of Samples to be Analyzed

One sample well per separation is dedicated to the ladder.

A 5 mL minimum volume in the tube is included.

Volume of Intercalating Dye

Volume of Separation

Gel

Volume of 1x Conditioning Solution

DNF-935 dsDNA Reagent Kit (1-1500 bp) Quick Guide for Fragment Analyzer

•

Follow the appropriate safety procedures such as wearing goggles, safety gloves and protective clothing.

5300/5400 system - 96 capillary; Ladder – well H12

Agilent dsDNA 935 (1-1500bp) DNF-935 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’

3.3.1. 5400 system; place in drawer “S”

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.

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

WARNING

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.

DNF-935 dsDNA Reagent Kit (1-1500 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-935-22 – DNA 1-1500 bp

3.2 DNF-935-33 – DNA 1-1500 bp

3.3 DNF-935-55 – DNA 1-1500 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.

100bp DNA Ladder result

Representative 100 bp DNA Ladder result double injected with 1 bp lower marker and 1,500 bp upper marker, using the Fragment Analyzer system with the DNF-935 dsDNA 935 Reagent kit. Method: DNF-935-22(“ultra-short” array).

DNF-935 dsDNA Reagent Kit (1-1500 bp) Quick Guide for Fragment Analyzer

Issue

Cause

Corrective Action

The peak signal is >> 20,000 RFU;

Input DNA sample concentration is

Further dilute input DNA sample

quantification accuracy.

Sample peak(s) migrate before or

Excess primer-dimer species in

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 915

930).

No peak observed for DNA sample

1 Sample concentration too low and out

1 Prepare more concentrated sample

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 DNF-935 dsDNA 935 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 1 bp Lower Marker.

migrate with 1,500 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.

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, 35 bp – 5,000 bp (DNF-

915), dsDNA 920 Reagent kit, 75 bp – 15,000 bp (DNF-920), or dsDNA 930 Reagent kit, 75 bp –20,000 bp (DNF-

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 and

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

DNF-935 dsDNA Reagent Kit (1-1500 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

Edition 10/20

SD-AT000125

Agilent DNF-935 Quick Start Guide

Specifications and Main Features

Frequently Asked Questions

User Manual

Specifications

Kit Components – 500 Sample Kit

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.

Technical Support and Further Information