Agilent DNF-464 HS Quick Start Guide

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

Results using DNA Fragment standards at 600 pg/µL and DNA smears at 1 ng/µL prepared from 1x TE buffer.

Results using DNA Fragment standards and DNA smears prepared from 1x TE buffer.

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%

DNF-464 HS Large Fragment Quick Guide for Fragment Analyzer Systems

WARNING

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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

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.

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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