Agilent Genomic DNA Quick Start Guide

Analytical specifications1

Genomic DNA 165Kb assay

Sizing Range

1.3 kb – 165 kb

Sizing Accuracy

+ 15%

Sizing Precision

20% CV

DNA Fragment Concentration Range

0.3 pg/µL - 30 pg/µL input DNA

DNA Smear Concentration Range

5 pg/µL - 500 pg/µL input DNA

gDNA Quantification Precision

25% CV

Maximum gDNA Concentration

500 pg/µL

Physical Specifications

Total electrophoresis run time

70 minutes (Fast Method), 180 minutes (Extended Method)

Samples per run

12-Capillary: 11 (+1 Ladder Well)

Sample volume required

2 µL

Kit stability

4 months

Agilent Genomic DNA 165 kb Kit
Quick Guide for Femto Pulse
System

The Agilent Femto Pulse system is an automated capillary electrophoresis platform for scalable,
flexible, fast, and reliable electrophoresis of nucleic acids.

This Quick Guide is intended for use with the Agilent Femto Pulse system only. The Genomic DNA 165
kb kit (275 Samples) (Part # FP-1002-0275) is designed for the pulsed-field CE separation, sizing and
quantitation of high molecular weight, low concentration DNA smears and/or fragments from 1.3 kb
though 165 kb.

Specifications

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Result based on Lambda DNA Fragment.

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FP-1002 Genomic DNA 165 kb Quick Guide for Femto Pulse Systems

DNF-498-0012

Dilution Buffer 0.25x TE, 12mL

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DNF-497-0060

0.25x TE Rinse Buffer, 60mL

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GP-435-0100

Storage Solution, 100 mL

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*Not Orderable.

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Kit Components – 275 Sample Kit

Kit Component
Number

Part Number
(Re-order Number)

Description Quantity Per Kit

5191-6604*

FP-1002-FR*

5191-6617*

Femto Pulse, 4oC

FP-5001-0250 FP Large DNA Separation Gel, 250 mL 1

DNF-306-0005 BF-P25 Blank Solution, 5 mL 1

DNF-325-0075 5x Inlet Buffer, 75 mL 1

Genomic DNA 165 kb, FR

FP-6001-U030

FP-8001-0003

FP-7002-U035 FP 165 kb Ladder, 35 μL 1

C27-130 Eppendorf LoBind 0.5 mLTubes (Bag of 50) 1

C280-101 Wide-Bore Genomic Pipette Tips, 1 Box 1

DNF-425-0050 5x Conditioning Solution, 50 mL 1

FP Intercalating Dye, 30 μL

FP gDNA Diluent Marker, 3 mL

Genomic DNA 165 kb, 275, RT

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WARNING

Refer to product safety data sheets for further information

When working with the Femto Pulse assay follow the appropriate safety procedures such as
wearing goggles, safety gloves and protective clothing.

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FP-1002 Genomic DNA 165 kb Quick Guide for Femto Pulse Systems

Environmental

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

Additional Material Required for Analysis with the Femto Pulse System

• Femto Pulse system with LED fluorescence detection:

• Femto Pulse system (p/n M5330AA)

• FP 12-Capillary Array, 22 cm (p/n A1600-1250-2240)

• Agilent Femto Pulse controller software (Version 1.0 or higher)

• Agilent ProSize Data Analysis software (Version 3.0 or higher)

Additional equipment/reagents required (not supplied)

• 96-well PCR sample plates. Please refer to Appendix – Femto Pulse Compatible Plates and Tubes in the Femto Pulse
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

• Wide-Bore Genomic pipette tips, Thermo Scientific #21-402-157, as needed for pipetting gDNA samples

• 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 (Femto Pulse system): BD Falcon #352070, available from Fisher Scientific #14-432-22 or VWR #21008-940

• 250 mL (Femto Pulse system or larger volumes): Corning #430776, available from Fisher Scientific #05-538-53 or

VWR #21008-771

• Capillary Storage Solution (p/n GP-440-0100)

Essential Measurement Practices

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

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FP-1002 Genomic DNA 165 kb Quick Guide for Femto Pulse Systems

Marker/Ladder/Sample Preparation

General Information

1. The recommended 96-well sample plate for use with the Femto Pulse system is a semi-skirted PCR plate from

Eppendorf (#951020303). Please refer to the Appendix – Femto Pulse Compatible Plates and Tubes in the Femto
Pulse User Manual for a complete approved sample plate list. The system has been designed to operate using
these dimensions/styles of PCR plates.

2. Store the gDNA Diluent Marker solution and the 165 kb Ladder at -20°C upon arrival.

3. Allow the marker and ladder solution to warm up to room temperature prior to use. Briefly spin the tubes after

thawing to ensure liquid is at the bottom of the tube.

NOTE: The use of PCR plates with different dimensions to the above recommended plate could lead to decreased injection quality and consistency. Damage to the capillary array cartridge tips is also possible.

165kb Ladder Handling and Storage

Prior to first use, the 165 kb Ladder should be aliquoted to minimize the number of freeze/thaw cycles.

1. Equilibrate the 165 kb Ladder to room temperature for about 30 min and mix by gently pipetting up-and-down up to

3 times with wide-bore genomic pipette tips (provided with kit) and a pipettor set to a 15 μL volume. Do not vortex
of flick the Ladder tube. Only wide-bore genomic pipette tips should be used to mix the 165 kb Ladder solution.

2. Aliquot the 165 kb Ladder solution into 7 aliquots, 5 μL each, using the provided wide-bore genomic tips and the

provided Eppendorf LoBind 0.5 mL tubes. Each aliquot can be used up to 4 times (1 μL per use).

3. Store the 165 kb Ladder aliquots at -20°C. Store the in-use vial of the 165 kb Ladder at 2-8°C for up to 2 weeks.

Avoid freeze-thawing of the 165 kb Ladder more than 4 times (additional freeze-thawing may result in degradation
of the higher molecular weight fragments in the 165 kb Ladder).

165 kb Ladder Working Solution

1. Before use, equilibrate the 165 kb Ladder aliquot to room temperature for about 30 min.

2. In an Eppendorf LoBind 0.5 mL tube (provided), aliquot 9 μL of the DNF-498 0.25x TE Dilution Buffer.

3. To the same Eppendorf tube aliquot 90 μL of the FP-8001 gDNA Diluent Marker solution. Mix the contents of the

tube by vortexing.

4. Mix the 165 kb Ladder aliquot very slowly by pipetting 1-2 times with a wide-bore genomic pipette tip and a pipettor

set to ~5 μL volume.

5. Using a regular pipette tip, immediately aliquot 1 μL of the mixed 165 kb Ladder into the tube containing 99 μL of

the solution from steps 2 and 3 above (DNF-498 0.25x TE Dilution Buffer + FP-8001 gDNA Diluent Marker). Do not
Pipette up-and-down or vortex.

6. Using the wide-bore genomic pipette tip only and a pipettor set to a 20 μL volume slowly pipette the prepared 165

kb Ladder working solution up and down 5 times to mix. This is the 165 kb Ladder Working Solution; use within one
day of preparation.

7. Load 20 μL of the 165 kb Ladder Working Solution into Well 12 of a sample plate row that is to be analyzed.

8. The 165 kb Ladder should be run in parallel with the samples for each experiment. It is not recommended to import

a previously run 165 kb Ladder.

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