Agilent Genomic 55 kb BAC Quick Start Guide

Analytical specifications1

55 Kb BAC assay

Sizing Range

75 bp – 48,500 bp

Sizing Accuracy1

+ 15% or better

Sizing Precision1

15% CV

Single fragment at 48,500 bp1 3 pg/µL – 25 pg/µL input DNA

Maximum gDNA Concentration

25 pg/µL per fragment; 100 pg/µL total input DNA

Physical Specifications

Total electrophoresis run time

90 minutes

Samples per run

12-Capillary: 11 (+1 Ladder Well)

Sample volume required

2 µL

Kit stability

4 months

Agilent Genomic 55 kb BAC Kit Quick Guide for Femto Pulse Systems

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 55 kb BAC kit (275 Samples) (Part # FP-1003-0275) is designed for the Pulse-Field CE separation and sizing of bacterial artificial chromosome (BAC) fragments smaller than 55 kb in length.

Specifications

DNA Fragment Concentration Range

Lambda DNA sizing.

1kb and 1.5kb fragment sample sizing.

Single fragment at 1,000 – 2,000 bp2 1pg/µL – 12.5 pg/µL input DNA

FP-1003 55 kb BAC Quick Guide for Femto Pulse System

DNF-498-0012

Dilution Buffer 0.25x TE, 12mL

DNF-497-0060

0.25x TE Rinse Buffer, 60mL

GP-435-0100

Storage Solution, 100 mL

FS-SMO15

Mineral Oil Dropper Bottle, 15 mL

*Not Orderable.

•

Kit Components – 275 Sample Kit

Kit Component Number

Part Number (Re-order Number)

Description Quantity Per Kit

5191-6604*

FP-1003-FR*

5191-6618*

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

55 kb BAC, FR

FP-6001-U030

FP-8001-0003

FP-7003-U035 FP 55 kb BAC Ladder, 35 μL 1

C27-130 Eppendorf LoBind 0.5 mL Tubes (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

BAC, RT

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.

FP-1003 55 kb BAC Quick Guide for Femto Pulse System

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 systems 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 Part #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

FP-1003 55 kb BAC Quick Guide for Femto Pulse System

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 at -20ºC and the 55 kb BAC Ladder at 2-8ºC upon arrival.

3. Allow the gDNA Diluent marker and 55 kb ladder 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.

55 kb BAC Ladder Handling and Storage

Prior to first use, the 55 kb BAC Ladder should be aliquoted to minimize the number of freeze/thaw cycles.

1. Equilibrate the 55 kb BAC Ladder to room temperature for about 30 min and mix briefly by vortexing. Aliquot 5.0 µL

of the 55 kb BAC Ladder to 7 different Eppendorf LoBind tubes (provided with kit) using the provided wide-bore genomic pipette tips. Each aliquot is good for 4-times use (1 µL per use).

2. Store the 55 kb BAC Ladder aliquots at 2-8ºC; avoid freeze-thawing.

55 kb BAC 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.

4. Aliquot 1 μL of the 55 kb BAC Ladder into the same Eppendorf tube containing 99 μL of the solutions from steps 2

and 3 above (FP-8001 Diluent Marker + 0.25x TE). Vortex to mix. This is the 55kbBAC Ladder Working Solution; use within one day of preparation.

5. Load 20 μL of the prepared 55 kb BAC Ladder Working Solution into Well 12 of a sample plate row that is to be

analyzed.

6. The 55 kb BAC Ladder should be run in parallel with the samples for each experiment. It is not recommended to

import a previously run 55 kb BAC Ladder.

NOTE: For samples that do not require predilution or the predilution is less than 160x, the sample matrix is expected to

influence sample sizing. Please refer to Preparation of 55 kb BAC Ladder Working Solution for instructions on preparing a 55 kb BAC Ladder Working Solution that contains the sample matrix.

Sample Plate Preparation

Important gDNA Sampling Procedures:

Before sampling, the sample stock of BAC fragments must be acclimatized to room temperature for at least 30 minutes.

1. The total input BAC sample concentration should be no higher than 100 pg/μL. As a general note, smaller BAC

fragments should be run at a lower limit of the concentration range; larger BAC fragments should be run at a higher limit of the concentration range.

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