May 28, 2026

Microbial cfDNA sample processing and extraction protocol with the QIAamp MinElute ccfDNA Kit

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l6oknklqe/v1
  • 1Statens Serum Institut
Martin Schou Pedersen
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6oknklqe/v1
Protocol Citation: Martin Schou Pedersen 2026. Microbial cfDNA sample processing and extraction protocol with the QIAamp MinElute ccfDNA Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6oknklqe/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 27, 2026
Last Modified: May 28, 2026
Protocol  Integer ID: 318000
Keywords: cfDNA, cell free DNA, mcfDNA, microbial, QIAamp MinElute ccfDNA Mini Kit, Streck, microbial cfdna sample processing, based cfdna purification, quality cfdna suitable for downstream oxford nanopore promethion, cfdna purification, microbial cfdna, extraction protocol with the qiaamp minelute ccfdna kit, quality cfdna, µl of cfdna, cfdna, qiaamp minelute ccfdna kit, free dna bct tube, free dna bct tubes via sequential centrifugation, downstream oxford nanopore promethion, free dna, plasma isolation from whole blood, extraction of cell, extraction protocol, purification, plasma isolation, human plasma
Abstract
This protocol describes standardized procedures for the collection, processing, and extraction of cell-free DNA (cfDNA) from human plasma, developed for microbial cfDNA sequencing. The goal is to obtain high-quality cfDNA suitable for downstream Oxford Nanopore PromethION sequencing.
The workflow covers two stages: plasma isolation from whole blood collected in Streck Cell-Free DNA BCT tubes via sequential centrifugation, followed by magnetic bead-based cfDNA purification using the QIAamp MinElute ccfDNA Kit. Approximately 50 µL of cfDNA is eluted per sample, with an expected yield of 15–40 ng as quantified by Qubit fluorometry.
Guidelines
This method prepares microbial cfDNA that can be prepared for sequencing in the following protocol:
Martin Schou Pedersen 2026. Microbial cfDNA Library Preparation and Sequencing with Oxford Nanopore. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v99e84v3e/v1
Materials
ABC
MaterialCatalog No.Supplier
Cell-Free DNA BCT tubes (Streck)218997Streck Inc., La Vista NE, USA
LoBind DNA 2 mL tubes525-0131VWR
QIAamp MinElute ccfDNA Mini Kit (50)55204Qiagen
Qubit dsDNA HS Assay KitQ33231Thermo Fisher
Qubit Flex Assay Tube StripsQ33252Thermo Fisher
DynaMag-2 magnet12321DThermo Fisher
CAPPRondo VORTEX mixerCRV-45XDACOS
CAPPRondo High Speed MinicentrifugeCR-1512DACOS
Eppendorf Thermomixer5382000015Eppendorf
Safety warnings
Please comply with local rules and SOP for handling the reagents and materials in this protocol.
Ethics statement
Human plasma collection for this protocol requires prior approval by the users' Institutional Ethics Board or equivalent ethics committee.
Before start
Note Streck Cell-Free DNA BCT tubes protect cfDNA for up to 14 days at room temperature. 

All reagents should be stored according to kit instructions.

The method has been changed to accomodate the limitations of the Mini kit og the use of a microcentrifuge.

Always include a negative water control.

Extracted cfDNA can be stored at −80°C and processed in bulk when ready.
Plasma Preparation
This starting point is based on the assumption that samples arrive in in Streck Cell-Free DNA BCT tubes.
Centrifuge the sample tubes at 1,600 × g (RCF) for 20 minutes.
Pipette plasma into 2 mL LoBind tubes, maximum 1 mL per tube.
Centrifuge at 15,595 × g (RCF) for 10 minutes.

Note
Adding more than 1 mL per tube will result in suboptimal pelleting of cells.

Allocate 3 mL plasma per sample for extraction.
Pipette residual plasma (not needed for extraction) into 2 mL LoBind tubes to store in −80°C freezer.

Note
Expected volume: 4–6 mL plasma per Streck tube.

cfDNA Extraction (QIAamp MinElute ccfDNA Kit)
For each sample, prepare 2 LoBind tubes each containing:
1.5 mL Plasma
45 µL Magnetic Bead Suspension
82 µL Proteinase K
225 µL Bead Binding Buffer

Incubate tubes at room temperature (15–25°C) for 10 minutes with continuous mixing (invert/tilt to prevent beads from settling).
Centrifuge tubes at 200 × g for 30 seconds to collect liquid from the cap.
Place one tube per patient on the magnet for >1 minute until liquid is clear. Remove supernatant.
Add the liquid from the second tube for the same patient to the emptied tube on the magnet.
Wait 1 minute until the liquid is clear.
Remove supernatant.

Remove tube from magnet.
Add 200 µL Bead Elution Buffer and mix by inversion.
Transfer contents to a new Bead Elution tube.
Place on Thermomixer at 300 rpm for 5 minutes at room temperature.
Place the tube on the magnet for >1 minute until liquid is clear.
Transfer supernatant to a new Bead Elution tube.
Note
Turn on the Thermomixer and set to 56°C now so it is ready for later.

Add 300 µL Buffer ACB to the tube.
Vortex for 10 seconds.
Centrifuge briefly to remove liquid from cap.
Prepare one column per sample with a 2 mL collection tube.
Load the supernatant onto column.
Centrifuge at 6,000 × g for 1 minute.
Transfer column to a new 2 mL collection tube.
Add 500 µL Buffer ACW2 to column.
Centrifuge at 6,000 × g for 1 minute.
Transfer the column to a new 2 mL collection tube.
Centrifuge at 15,595 × g for 3 minutes.
Transfer the column to a new 1.5 mL Bead Elution tube.
Incubate on a Thermomixer at 56°C for 3 minutes with the cap open.
Add 50 µL nuclease-free water to the center of the column membrane.
Incubate for 1 minute at room temperature.
Centrifuge at 15,595 × g for 1 minute.
Return the 50 µL eluate to the center of the column membrane.
Incubate for 1 minute at room temperature.
Centrifuge at 15,595 × g for 1 minute.
Collect the eluate (~50 µL).
Measure the DNA concentration with Qubit.
Expected yield: 15–40 ng total.

Note
For guidance please refer to:
Josh Quick 2025. DNA Quantification using the Qubit Fluorometer. protocols.io https://dx.doi.org/10.17504/protocols.io.261gekdeyg47/v1

Store the cfDNA on ice until ready library preparation.
If not proceeding to library prep on the same day, store cfDNA at −80°C.
Next Step: Library Preparation and Sequencing
After completing cfDNA extraction and quality measurement (Qubit), continue with the following protocol for end repair, barcode ligation, adaptor ligation, flow cell loading, and sequencing

Martin Schou Pedersen 2026. Microbial cfDNA Library Preparation and Sequencing with Oxford Nanopore. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v99e84v3e/v1