Jun 03, 2026

1. Nanobind® HT HMW DNA extraction – Bacteria KingFisher Apex

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvme79og3p/v1
  • 1CFSAN/FDA;
  • 2USDA;
  • 3US FDA
Jae Hee Jang
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvme79og3p/v1
Protocol Citation: Jae Hee Jang, Ellie Meeks, Kathryn Judy, Maria Hoffmann 2026. 1. Nanobind® HT HMW DNA extraction – Bacteria KingFisher Apex. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvme79og3p/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: March 29, 2024
Last Modified: June 03, 2026
Protocol  Integer ID: 97536
Keywords: Nanobind, HT CBB kit, PacBio, Pacific Biosciences, HMW DNA extraction, bacterial DNA, automated hmw dna extraction, workflow for automated hmw dna extraction, hmw dna, bacteria kingfisher apex this procedure, bacteria kingfisher apex, thermo fisher kingfisher apex system, positive bacteria, bacteria, kingfisher
Abstract
This procedure describes the workflow for automated HMW DNA extraction from cultured Gram-negative and Gram-positive bacteria on the Thermo Fisher KingFisher Apex system. This protocol uses the KingFisher 96 deep-well magnetic head, 96 deep-well plates, and 96 deep-well tip comb.
Guidelines
The Nanobind HT CBB kit has enough reagents for 96 extractions to be run in one of the following formats: 1 run x 96 samples, 2 runs x 48 samples, or 4 runs x 24 samples. We do not recommend running fewer than 24 samples per run as the kit is designed to accommodate dead volumes for a maximum of 4 runs (4 runs x 24 samples).

Input requirements:
Amount: 5x108 Gram-negative or Gram-positive bacteria
  • This protocol has been validated on Gram-negative bacteria including E. coli, S. enterica, S. sonnei, K. pneumoniae, and P. aeruginosa, and Gram-positive bacteria including L. monocytogenes, E. faecalis, and S. aureus.
  • No systematic difference has been observed in DNA QC or sequencing results between fresh and frozen cells.

Extracted DNA can be stored in Buffer EB at 4°C for several months. Long term storage at −20°C or −80°C can be used if necessary. Avoid freeze/thaw cycles since this can degrade high molecular weight DNA
Materials
Reagents
  • Nanobind HT CBB Kit (PacBio Cat# 102-762-700)
  • If new, 100% Ethanol, Molecular Biology Grade (Thermo Scientific Cat# BP2818500 or equivalent)
  • 1x PBS (Thermo Fisher Scientific Cat# 10010023 or equivalent)
  • STET buffer (Cepham Life Sciences Cat# 10414-0 or equivalent)
  • Lysozyme (MP Biomedicals Cat# 100831 or equivalent)
  • for Gram-positive samples, either
  • Lysostaphin (Sigma Aldrich Cat# L7386)
  • or MetaPolyzyme Multilytic Enzyme Mix (Sigma Aldrich Cat# MAC4LDF)
  • Isopropanol, Molecular Biology Grade (Fisher Scientific Cat#17140576 or equivalent)
  • Qubit 1x dsDNA Broad Range Kit (Q33266 or Q33265)
  • Genomic DNA 165 kb Kit (Agilent Cat# FP-1002-0275)

Supplies
  • Semi-micro cuvettes (Greiner Bio-One Cat# 613101 or equivalent)
  • Serological Pipet, 1 mL, Individually Packaged Sterile (Corning Cat# 357521 or equivalent)
  • KingFisher Apex 96 Deep-Well Plates, Barcoded (Thermo Fisher Scientific Cat# 95040450B)
  • KingFisher Apex 96 Deep-Well Tip Combs, Barcoded (Thermo Fisher Scientific Cat# 97002534B)
  • Micropipette Tips, Sterile, Filtered (P20, P200, P1000), any manufacturer
  • Wide-bore Pipette Tips, Sterile, Filtered (P200) (Mettler Toledo Cat# 30389241 or equivalent)
  • Reagent Reservoirs, (Thermo Fisher Scientific Cat# 13681504 or equivalent)
  • 1.5 mL Protein LoBind Tubes (Eppendorf Cat# 022431081)
  • 2.0 mL DNA LoBind Tubes (Eppendorf Cat# 022431048)
  • Qubit Assay Tubes (Thermo Fisher Scientific Cat# Q33252)

Equipment
  • Serological Pipette Controller, any manufacturer
  • Micropipettes, standard (P2, P20, P200) and multichannel (P200, P1000), any manufacturer
  • KingFisher Apex System with 96 Deep-Well Head (Thermo Scientific Cat# 5400930)
  • with installed KingFisher Apex 96 Deep-Well Heating Block (Thermo Scientific Cat# 24075930)
  • UV/Vis Spectrophotometer (Pharmacia Biotech UV/Vis-Spectrophotometer Ultrospec 1000 or equivalent)
  • Microcentrifuge (Eppendorf Cat# 5406000240 or equivalent)
  • Vortex (Benchmark Scientific Cat# BV101-B or equivalent)
  • Heating block (Eppendorf Cat# 2231001127 or equivalent
  • Qubit Fluorometer (4.0, 3.0, or 2.0) (Thermo Fisher Scientific Cat# Q33238)
  • DNA-compatible Spectrophotometer (Thermo Fisher Scientific Cat# NDLPLUSGL or equivalent)
  • Femto Pulse System (Agilent Cat# M5330AA)

Safety warnings
Warning: cell inputs that yield >25 μg DNA may result in Nanobind disks being “dropped” in the Lysis/Binding solution and/or cause well-to-well contamination.

Chemical 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 

Nanobind HT CBB Kit: See PacBio SDSs for addition information. Take proper precautions and wear appropriate PPE when handling reagents 
- Proteinase K: GHS Category 3 for skin irritation, Category 1 for respiratory sensitizer 
- Buffer CLE3: GHS Category 3 for skin irritation, Category 2 for eye irritation, Category 3 for acute aquatic toxicity 
- Buffer BL3: GHS Category 4 for acute oral toxicity and acute inhalation toxicity, Category 2 for skin irritation and 2a for eye irritation, Category 3 for acute aquatic toxicity 
- Buffer CW1: GHS Category 3 for skin irritation, acute aquatic toxicity, and chronic aquatic toxicity 
Before start
Buffer CW1 and CW2 are supplied as concentrates. This kit uses CW1 with a 60% final ethanol concentration. This kit uses CW2 with a 60% final ethanol concentration. Before using, add the appropriate amount of ethanol (96–100%) to Buffer CW1 and Buffer CW2 as indicated on the bottles.

Be sure to measure the density of cultured cells to ensure that there is no overloading or underloading of the reactions. Overloading and underloading can lead to suboptimal DNA yields.
Before Starting
If first time using Nanobind HT HMW DNA Extraction kit:
  • Add the appropriate amount of molecular biology grade ethanol (96 – 100%) to buffers CW1 and CW2 before first use. Mark the bottles to indicate the date ethanol was added 
Prepare sufficient volume STET buffer + Lysozyme for processing all samples to be extracted. Scale the values below based on the number of samples:
Reagent8 samples
STET buffer 500 uL
Lysozyme 0.005 g
Figure 1: This concentration mix is for one column of samples. Increase amounts of reagents in accordance with the ratio provided if there are more than 8 samples.
After adding the lysozyme, incubate at 37 °C for 10 – 15 minutes until fully dissolved. STET buffer + Lysozyme mix should used on the day of preparation
Plate preparation
Collect 7 KingFisher 96 deep-well plates and prepare the following:
Nanobind storage plate:
  • Place one 3 mm Nanobind Disk per well.

Nanobind disks do not need to be perfectly centered in the wells, but ensure they are at the bottom of the well and not stuck to the side walls.
Elution plate:
  • 100 µL of Buffer EB per well
Wash plate 1:
  • 700 µL of Buffer CW1
Wash plate 2:
  • 700 µL of Buffer CW2
Wash plate 3:
  • 700 µL of Buffer CW2
Tip plate:
  • Insert one KingFisher Apex 96 deep-well tip comb into the plate
Turn on the Kingfisher Apex, select the Bacteria_Nanobind_HT_APEX script, and press ‘Start’. Insert plates into the KingFisher Apex instrument as indicated on the display and press ‘Next’ after every plate to confirm position. The seventh plate, the Lysis/Binding plate, should be prepared after all other plates are ready
Cell Harvesting
1m
Measure optical density (OD) of broth culture for each sample. The instructions in this section apply to the Pharmacia Biotech Ultrospec 1000. Follow manufacturer's instructions for other spectrophotometer models
Turn on spectrophotometer using the rear switch
Verify the wavelength is set to 600 nm
Ensure the compartment is empty, close the lid, and press F2 to calibrate
Fill a cuvette with 750 µL broth blank and insert into the compartment with the arrow facing the back of the instrument

Close the lid and press the blank button at the bottom center of the keypad
After blanking the instrument, remove and save the blank cuvette and insert the first sample cuvette
Close the lid and wait for the OD600 measurement to stabilize. Record the stabilized value as the OD600 for the sample, then remove the sample
Measure OD600 for all samples, repeating the blank measurement after every few sample measurements. OD readings increase as the instrument warms up, so it is important to blank regularly
Pellet cells based on OD600 measurement:
Scale input such that the cells pelleted will yield 5 µg - 25 µg DNA. Input should be 5x108 Gram-negative or Gram-positive bacteria, or 0.5 mL of 1 OD600 broth culture
To calculate μL to pellet, use the formula . Very low OD600 measurements (<0.35) will require multiple pellets to gather sufficient cells. Always round up μL to pellet to ensure sufficient cell density:
  • If the calculated volume is 325 µL , pellet 350 µL
  • If the calculated volume is 350 µL , pellet 400 µL
Fill a 1.5 mL protein Lo-bind tube with the rounded volume of broth culture. Do not use the culture aliquoted into the cuvette for OD600 measurement
Centrifuge at 16000 x g, 4°C, 00:01:00 to pellet cells. After pelleting, remove supernatant with a pipette

1m
Optionally, pelleted cells can be frozen at -20 °C for up to 3 months before continuing

Sample and Lysis/Binding Plate Preparation
Add 10 µL of 1x PBS to fresh or frozen (and briefly thawed) pelleted cells
  • Mix until cell pellet is fully resuspended without visible lumps.
Pipette mix 10x with a standard P200 pipette to resuspend cells, or until the cell pellet is fully resuspended without visible lumps
  • Sticky cell types may require additional pipette mixing or vortexing
  • Additional mixing at this step will not affect DNA size. However, incomplete resuspension will result in inefficient lysis and digestion which will lead to low yield, low purity, and high heterogeneity
Add 50 µL of STET Buffer + lysozyme and pulse vortex for 1s x 10 times (max setting)
  • For some Gram-positive bacteria, such as S. aureus, lysostaphin or MetaPolyzyme should be used as a supplement. Add 2.5 µL reconstituted lysostaphin or MetaPolyZyme to 50 µL STET buffer + lysozyme per extraction
Add the samples prepared in Steps 7 - 9 to individual wells of the Lysis/Bidning KingFisher deepwell plate
Add the following reagents in the order listed below. Add reagents directly to the liquid, not against the side of the well
  • Add 10 µL of Proteinase K to each well.
  • Add 5 µL of Buffer CLE3 to each well.
  • Add 20 µL of RNase A to each well.

Note: the sample and reagents MUST be added to the wells in the order described above
Ensure the instrument is set up with the 96 deep-well magnetic head and the 96 deep-well heating block
Finally, insert the Lysis/Binding plate into the Kingfisher Apex instrument. The protocol will start when the final plate is loaded and the ‘Next’ button is pressed
During the automated protocol
1h 41m
When prompted by the instrument (~00:35:00 after start), remove the Lysis/Binding plate (Plate 1) from the instrument and add 100 µL of Buffer BL3 to each well. Re-insert the plate and press ‘Next’ to resume the protocol

Note: add BL3 gently against the side of the well into the Lysis/Binding solution. Adding BL3 directly to the Lysis/Binding solution may affect extraction performance
35m
When prompted by the instrument (~00:56:00 after start, ~00:22:00 after last addition), remove the Lysis/Binding plate (Plate 1) from the instrument and add 250 µL of isopropanol to each well. Re-insert the plate and press ‘Next’ to resume the protocol

Note: add isopropanol gently against the side of the well into the Lysis/Binding solution. Adding isopropanol directly to the Lysis/Binding solution may affect extraction purity
22m
At the end of the run (~01:40:00 after start, ~ 00:44:00 after last addition), remove plates as indicated on the instrument display. The protocol will end when the final plate is removed and the ‘Next’ button is pressed

44m
Post-Automated Protocol
12h
When the instrument is empty and the door is closed, run 00:15:00 UV from the KingFisher instrument menu to decontaminate. Do not start UV with samples present in the instrument

Transfer eluates from the Elution Plate (Plate 6) to a new storage plate or storage tubes.
  • The protocol is designed to leave the Nanobind disks in the Elution Plate. On occasion, the disk may be transferred back to the tip comb storage plate (Plate 7) after elution. This does not affect extraction performance
  • When transferring eluate, a small amount of liquid may remain on the Nanobind disk. Use a P200 pipette tip to transfer any liquid remaining on the Nanobind disk
Pipette-mix the sample 10 times with a standard P200 pipette to homogenize and disrupt any unsolubilized “jellies” that may be present
  • Take care to disrupt any regions that feel more viscous than other regions
  • Limited pipette mixing will not noticeably reduce DNA size or sequencing read lengths but is important for accurate quantitation and consistent sequencing performance
Let eluate rest Overnight at Room temperature to allow DNA to solubilize
  • Visible “jellies” should disperse after resting
12h
Following overnight rest, pipette mix 10 times with a standard P200 pipette and analyze the recovery and purity
Sample QC
Perform a NanoDrop UV/VIS measurement to determine total nucleic acid concentration as well as purity (A260/A280, A260/A230)
  • 260/280 ratios should consistently be 1.7–2.0
  • 260/230 ratio can vary from 1.2–1.8
  • Samples with UV purities within the expected range should sequence well. UV purities outside of these ranges may indicate abnormalities in the extraction process
Perform a Qubit dsDNA BR assay measurement to determine DNA concentration
  • We recommend the Qubit 3.0 (Thermo Fisher Scientific) with the dsDNA BR assay kit. The dsDNA HS assay kit is not recommended as the concentration measurements may be unreliable
Use the Agilent Femto Pulse Genomic DNA 165 kb Kit for HMW DNA size QC. This step should always be performed when working with novel sample types but is otherwise optional. See the corresponding SOP for details
  • It is recommended to dilute the unsheared genomic DNA to 500 pg/µL . Finger tap to mix
  • Avoid mixing with a standard pipette. This will shear the DNA. Always use a wide-bore pipette tip when making dilutions
  • The mode of extracted cultured Gram-negative and Gram-positive bacteria DNA measured on the Femto Pulse system (Agilent Technologies) is typically 100 kb+