Jun 03, 2026

Using the Agilent Genomic DNA 165 kb Kit on the Femto Pulse System

DOI
https://dx.doi.org/10.17504/protocols.io.ewov19oeplr2/v1
  • 1CFSAN/FDA;
  • 2USDA;
  • 3US FDA
Jae Hee Jang
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.ewov19oeplr2/v1
Protocol Citation: Jae Hee Jang, Ellie Meeks, Kathryn Judy, Maria Hoffmann 2026. Using the Agilent Genomic DNA 165 kb Kit on the Femto Pulse System. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov19oeplr2/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 21, 2024
Last Modified: June 03, 2026
Protocol  Integer ID: 100221
Keywords: Femto Pulse, gDNA, HMW, quantification, sizing, extracted hmw dna, quantification of hmw dna, agilent genomic dna 165 kb kit, hmw dna, pacbio sequencing, agilent genomic dna, sheared dna, femto pulse system this procedure, effectiveness of dna shearing, dna shearing, femto pulse system, gdna, traditional pulse field gel electrophoresi
Abstract
This procedure outlines the quantification of HMW DNA using the Agilent Genomic DNA 165 Kb Kit on the Femto Pulse System. It performs sizing of gDNA up to 165 kb much more rapidly than traditional pulse field gel electrophoresis. It can be used to assess quality of extracted HMW DNA, effectiveness of DNA shearing, and size of final libraries for PacBio sequencing.

Expected results vary based on the input sample type (gDNA, sheared DNA, prepared library pool).
Materials
Reagents
  • Agilent Genomic DNA 165 kB kit (Agilent Cat# FP-1002-0275)
  • UltraPure Distilled Water (DNase, RNase-Free) (Invitrogen Cat# 10977015 or equivalent)

Supplies
  • Pipette Tips, Sterile, Filtered (P20, P200, P1000), any manufacturer
  • Wide-Bore Pipette Tips, Sterile, Filtered (P200) (Mettler Toledo Cat# 30389241 or equivalent)
  • 50 mL Falcon tubes (Corning Cat# 352098 or equivalent)
  • Eppendorf LoBind 1.5 mL tubes (Eppendorf Cat#022431021)
  • 96 DeepWell Plate 1 mL (Fisher Scientific Cat# 12-566-120)
  • Eppendorf PCR Lobind Plate 96 well plates (Eppendorf Cat# 0030129504)

Equipment
  • Femto Pulse System (Agilent Cat# M5330AA)
  • Micropipettes, single (P2, P20, P200, P1000) and multichannel (P200, P1000), any manufacturer
  • Microcentrifuge (Benchmark Scientific Cat# C1012 or equivalent)
  • Vortex (Benchmark Scientific Cat# BV101-B or equivalent)
Safety warnings

Safety information
Chemical Safety Warning: Take proper precautions and wear appropriate PPE when handling potentially hazardous chemicals. Ensure that chemicals, spent containers, and unused contents are disposed of in accordance with governmental safety standards

Agilent Genomic DNA 165 Kb Kit: See Agilent SDSs for additional information. Take proper precautions and wear appropriate PPE when handling reagents
5x Inlet Buffer: GHS Category 2 for skin corrosion/irritation and serious eye damage/eye irritation, Category 1 for skin sensitization and 1B for reproductive toxicity, Category 3 for specific target organ toxicity - single exposure (respiratory tract irritation). Contains triethylamine, boric acid, and 2-methyl-2H-isothiazol-3-one
5x Conditioning Solution: GHS Category 1 for skin sensitization. Contains 2-methyl-2H-isothiazol-3-one
FP large Separation Gel: GHS Category 1 for skin sensitization. Contains 2-methyl-2H-isothiazol-3-one
FP Intercalating Dye: GHS Category 4 for flammable liquids, category 2B for eye irritation. Contains dimethyl sulfoxide
Storage Solution: GHS Category 2B for eye irritation, Category 1 for skin sensitization. Contains 2-methyl-2H-isothiazol-3-one

Sample preparation
Dilute samples to appropriate concentrations using distilled, nuclease-free water or 0.25x TE dilution buffer. Use the same diluent for all samples within a run
  • Use 500 pg/µL for samples expected to have a wider distribution of various fragment sizes (e.g., HMW genomic DNA)
  • Use 250 pg/µL for samples expected to have a more uniform size distribution (e.g., sheared DNA, DNA libraries)

Solution tube preparation
Check to see if the current solution levels shown on the Femto Pulse application are adequate. Solution levels shown on the Femto Pulse are not anutomatically detected and are based off initial input from the user. Confirm solution levels by visually inspecting the tubes.The following table lists the Femto Pulse system volume specifications

Figure 1: Depending on the number of samples, a certain volume of intercalating dye, separation gel, and 1x Conditioning solution are needed. For every 10 samples, add another 1.0 µL to the initial 1.0 µL intercalating dye and add another 10 mL to initial 10 mL Separation gel and 1x Conditioning Solution. There should be a maximum of 10 samples per row, as 2 spots are needed per row for ladders

If the volumes listed are inadequate:
For the tube of FP Large DNA Separation Gel:
  • Combine the appropriate amounts of Separation Gel and Intercalating Dye according to the table shown in Step 2 in a Falcon tube
  • Invert gently to mix

Note: Each mixture of FP Large DNA Separation Gel is good for 2-3 weeks. Prepared gel that is currently not in use can be stored at 4 °C . Make sure to protect prepared gel from light
For the tube of 1x Conditioning Solution:
  • Combine 40 mL of distilled, nuclease-free water with 10 mL of 5x Conditioning Solution in a Falcon tube and invert to mix
  • Using the freshly made 1x Conditioning Solution, fill the Conditioning Solution tube in the Femto Pulse system to the desired amount. Make sure to update the solution level on the Femto Pulse
  • Excess 1x Conditioning Solution can be stored at Room temperature

Reagent plate preparation
For Drawer B of the Femto Pulse system, prepare a 96 deep well plate as follows:
Row A: 1.0 mL of 1x Inlet Buffer per well. This row should be replaced with fresh buffer daily, or every new day that the Femto Pulse system is run

Note: the 165kb kit comes with 5x Inlet Buffer. Combine 40 mL of distilled water with 10 mL of the stock solution in a Falcon tube to make 1x Inlet Buffer
Row H: 1.0 mL of Capillary Storage Solution per well. This row of solution is good for one week, after which the whole plate should be replaced
For Drawer M of the Femto Pulse system, prepare a 96 well semi-skirted plate as follows:
Row A: 200 µL of 0.25x TE Rinse Buffer per well. This plate is to be replaced every new day that the Femto Pulse system is run
Place plates into their corresponding drawers
Diluent Marker and 165kb ladder prep
Divide new, unused tubes of Diluent Marker into aliquots of around 1 mL each, in 1.5 mL Eppendorf tubes. Use one tube at a time. Vortex to mix, then spin down

Note: Diluent Marker is temperature-sensitive, and exposure to Room temperature should be limited to one hour at a time
For new stock tubes of 165 kb ladder:
Thaw the tube slowly on ice then briefly spin down to collect all liquid at the bottom of the tube

Note: The 165 kb ladder is extremely sensitive! Do not flick, vortex, or pipette mix using regular pipette tips. The ladder should be handled using only the wide-bore pipette tips provided in the 165 kb kit
Using the provided wide-bore pipette tips, carefully mix the ladder up and down three times with the pipettor set to 20 µL

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)
Store the 165 kb Ladder aliquots at -20 °C
For aliquoted tubes of 165 kb ladder:
Thaw the tube slowly on ice. Briefly spin down to collect all liquid at the bottom of the tube. Do not flick, vortex, or pipette mix using regular pipette tips

Note: 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)
Sample plate preparation
Before starting:
  • Each row can hold up to 11 samples (well 12 should always contain the ladder). However, it is recommended to run more than one ladder per row so that if the ladder in well 12 does not look good, there is at least one back-up ladder to choose from. Loading a ladder from a separate file is not recommended
Using a clean 96-well sample plate, pipette 18 µL of FP-8001 gDNA Diluent Marker Solution (DM) to each well of the 96-well plate to contain a sample
Pipette 2 µL of each gDNA sample into the 18 µL of DM in the respective wells of the Sample Plate. Mix the sample wells by pipetting up and down three times with wide-bore genomic pipette tips
Prepare the 165 kb Ladder Working Solution:
In an Eppendorf LoBind 0.5 mL tube (provided), aliquot 9 µL of the DNF-498 0.25x TE Dilution Buffer
To the same Eppendorf tube aliquot 90 µL of the FP-8001 gDNA Diluent Marker solution. Mix the contents of the tube by vortexing
Mix the 165 kb Ladder aliquot very slowly by pipetting two times with a wide-bore genomic pipette tip and a pipettor set to 5 µL volume
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 13.1 and 13.2 above (DNF-498 0.25x TE Dilution Buffer + FP-8001 gDNA Diluent Marker). Do not pipette-mix or vortex
Using the wide-bore genomic pipette tip 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

Note: Use within one day of preparation
Load 20 µL of the 165 kb Ladder Working Solution into Well 12 (and other wells, if duplicates of the ladder are being run) of the sample plate row that is to be analyzed
Fill any unused wells within the row of the sample plate with 20 µL of BF-P25 Blank Solution

Note: The Blank Solution is light sensitive. Handle with care
After loading the samples and 165 kb Ladder Working Ladder in each well, check the wells of the sample plate to ensure there are no air bubbles trapped in the bottom of the wells. Centrifuge the plate to remove any trapped air bubbles. The presence of trapped air bubbles can lead to injection failures
Run the sample plate immediately once prepared, or cover the sample plate with a cover film, store at 2 °C to 8 °C , and use as soon as possible
To run the samples, place the plate in one of the three sample plate trays (Drawers 4-6 from the top, labelled 1, 2, and 3) of the Femto Pulse instrument
Starting the run
A 20 min Conditioning should be done at the start of each day a run is put on the Femto Pulse system. To start the conditioning process:
From the main screen of the Femto Pulse control software, select the Operation tab. Under the Capillary Array > Conditioning field press Add to queue. The Select Conditioning Method form will be displayed, enabling the user to select the conditioning method from the dropdown menu
Select the “20 min Conditioning” method from the dropdown menu. This method performs a 20 min conditioning solution flush followed by a 3 min Gel fill
Press OK to add the method to the instrument queue (press Cancel to abort adding the method)
Press the green Play icon to start the sequence loaded into the queue
Select Row, Group or Tray to run. Input names of samples, as well as location of ladders and blanks
Select Add to Queue, from the dropdown menus select the corresponding method based on your capillary length:
  • 3.1 FP-1002-22 – gDNA 165kb
  • 3.2 FP-1002E-22 – Extended gDNA 165kb (preferred)
Select OK to add method to the queue
Select the green Play icon to start the run
Analyzing the run
Ensure that the ladder for each row is properly labelled. An extended gDNA 165kb ladder should look like the following:

Figure 2: the ideal extended gDNA 165 kb ladder



Detected peaks can be added or deleted by right-clicking in the location of the correct / incorrect peak. Choose option "Accept change" on the pop-up menu to confirm the new peak location. After accepting the ladder, size information for the samples will be generated
Protocol references