Cell-Free DNA (cfDNA)
Purification Kit(version A3)
Catalog No. 25503
Catalog No. 25504

Copyright 2021 Active Motif, Inc.

Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of
any kind, either explicit or implied. Information may be changed or updated in this manual at anytime.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole or in part, without the prior written consent of Active Motif, Inc. This documentation is proprietary information and protected by the copyright laws of the United States and international
treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2018 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights
reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respective
companies.
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TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Cell-Free DNA (cfDNA) Purification Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Protocols
Buffer Preparation and Recommendations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Assay Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Section A. Experimental Set-Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Section B. Lysis/Binding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Section C. First Wash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Section D. Second Wash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Section E. Elution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

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Overview
Cell-free DNA can serve as a valuable source of information for non-invasive detection andmonitoring of various disease states. However, cell-free DNA is often found in limited amounts inplasma and serum samples. Active Motif’s Cell-Free DNA (cfDNA) Purification Kit provides a quick,simple method to isolate and concentrate lower molecular weight cell-free DNA from larger
nucleic acids within the sample.
The Cell-Free DNA (cfDNA) Purification Kit is designed to isolate cell-free DNA from humanplasma/serum samples. The fast, magnetic bead-based protocol isolates cell-free DNA from freshor frozen plasma that is suitable for use in downstream applications such as qPCR, ddPCR, DNA
methylation analysis, and Next generation sequencing (NGS).
The Cell-Free DNA (cfDNA) Purification Kit provides enough reagents to isolate cell-free DNA
from up to 100 ml or 250 ml human plasma. Easily process input volumes from 100 µl to 10 ml, orcombine with automation to streamline handling of multiple samples.

productformat

catalog no.

Cell-Free DNA (cfDNA) Purification Kit

100 ml
250 ml

25503
25504

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Kit Performance and Benefits
The Cell-Free DNA (cfDNA) Purification Kit can be used to isolate cell-free DNA from humanplasma samples. There are two package sizes for this kit, which enable isolation from up to 100ml and 250 ml human plasma respectively.
Input amounts: Both fresh and frozen plasma can be used with the Cell-Free DNA Purification Kit
in the range of 100 µl to 10 ml. However, fresh plasma tends to have higher yields.
Plasma from blood collected with Streck Cell-Free DNA BCT® Tubes must go through a Proteinase K treatment prior to cell-free DNA isolation to ensure optimal yields. Omitting the Proteinase
K treatment may decrease yields up to 50%.
Expected yield: The yield of cell-free DNA is approximately 1 - 100 ng per ml of plasma. Quantification by absorbance measurement (e.g. Nanodrop) may not be sensitive enough to accurately
determine yield. We recommend using the Qubit™ dsDNA High Sensitivity Assay to quantify
purified cfDNA.
Assay time: As little as 1.5 hours.

Cell-Free DNA (cfDNA) Purification Kit

Figure 1: TapeStation determination of isolated cfDNA size.
The Cell-Free DNA (cfDNA) Purification Kit was used to isolate cell-free DNA from 5 ml of healthy human plasma. Isolated DNA
was run on a TapeStation to assess the size of the purified DNA. Results show the majority of the purified samples is cell-free
DNA at the expected size of 150-200 bp.

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Figure 2: Reproducibility of the Cell-Free DNA (cfDNA) Purification Kit.
The Cell-Free DNA (cfDNA) Purification Kit was used to isolate DNA from 1 ml of plasma from four different human donors induplicate. The table above shows the total yield recovered from each sample with strong correlation between duplicates.

Figure 3: Automation compatibility with KingFisher™ Flex Purification System.
Both the Cell-Free DNA (cfDNA) Purification Kit and a competitor magnetic bead-based cfDNA purification kit (Competitor T)
were used to isolate DNA from 600 µl of plasma from a normal human donor in triplicate using the KingFisher Flex Purification System. Results show the total DNA yield (ng) per ml of plasma. The Cell-Free DNA (cfDNA) Purification Kit recovered
more DNA than the alternative purification method.

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3Kit Components and Storage
Cell-Free DNA Purification Kits are for research use only. Not for use in diagnostic procedures. Allcomponents are guaranteed stable for 6 months from date of receipt when stored properly.
Quantity
100 ml kit

Quantity
250 ml kit

Storage

Lysis/Binding Buffer AM2115 ml

3 x 95 ml

RT

Wash Buffer AM8

2 x 55 ml

5 x 55 ml

RTElution Buffer AM66 ml

15 ml

RT

2 x 1.33 ml

5 x 1.33 ml

RTReagents

Magnetic Bead Solution

Additional materials required but not supplied• Human plasma• Proteinase K (20 mg/ml) if plasma samples were from blood collected using Streck Cell-Free
DNA BCT® tubes• 20% SDS solution if plasma samples were from blood collected using Streck Cell-Free DNABCT® tubes• Pipettes and tips
• Vortex• Magnetic stand compatible for use with magnetic particles in microcentrifuge tubes.
Depending on plasma volume, magnetic stand compatible with 15 ml or 50 ml conical tubes
may also be used.
• 1.5 ml low retention microcentrifuge tubes (e.g. Eppendorf™ LoBind Microcentrifuge tubes)• Microcentrifuge• Fresh 100% ethanol

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Protocols
Buffer Preparation and Recommendations
Lysis/Binding Buffer AM2
The Lysis/Binding Buffer AM2 is shipped as a concentrate and requires the addition of 100%ethanol (EtOH) prior to use. Once ethanol is added, place a mark on the bottle to indicate it is acomplete solution and mix by inverting gently. If a precipitate is present, incubate the solution at37°C for 30 minutes. Close bottle tightly for long term storage and store at 4°C for up to one year.
100 ml Kit(per bottle)

250 ml Kit(per bottle)

Lysis/Binding concentrate

115 ml95 ml

100% EtOH

23 ml

19 ml

Wash Buffer AM8
Wash Buffer AM8 is shipped as a concentrate and requires the addition of 100% ethanol (EtOH)
prior to use. Once ethanol is added, place a mark on the bottle to indicate it is a complete solution and mix by inverting gently. If a precipitate is present, incubate the solution at 37°C for 30
minutes. Close bottle tightly for long term storage and store at 4°C for up to one year.
100 ml Kit(per bottle)

250 ml Kit(per bottle)

Wash Buffer concentrate

55 ml

55 ml

100% EtOH

51 ml

51 ml

80% EtOH
Prepare a fresh 80% ethanol (EtOH) solution prior to each extraction. Each sample will need aminimum of 2 ml 80% ethanol solution for washing steps.
Magnetic Bead Solution
Is provided ready to use. Mix beads prior to use. There should be no visible sedimentation at thebottom of the solution after mixing. Beads will settle quickly, so be sure to mix the magnetic beadsolution after adding to each sample. Failure to do so may result in inconsistent results.
Elution Buffer AM6
Is provided ready to use.

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5Assay Protocol
Read the entire protocol before use.
Prepare the reagents needed for the assay according to the instructions provided in the Buffer
Preparation and Recommendations protocol. Follow the instructions below to determine thevolume of reagents needed based on the amount of plasma used per sample.

Section A: Experimental Set-Up
The Cell-Free DNA (cfDNA) Purification Kit provides a protocol based on the amount of plasma
to be used for the purification. Any amount from 100 µl to 10 ml of fresh or frozen human plasma
can be used. Scale buffer and magnetic bead solution volumes accordingly using the tablebelow.

Plasma volume

Lysis/Binding
Buffer AM2

Magnetic BeadSolution

Tube sizex (x = ml of plasma)

1.25x

0.025x

N/A

0.5 ml625 µl12.5 µl

2 ml

1 ml

1.25 ml

25 µl

15 ml

5 ml

6.25 ml

125 µl15 ml or 50 ml*10 ml

12.5 ml

75 µl**

50 ml*Using a 50 ml tube for 5 ml or more of plasma is recommended over a 15 ml tube. While a 15 ml tube will work it may
lead to slightly lower yields.
**When working with 10 ml of plasma per tube, the recommended amount of Magnetic Bead Solution differs from therecommended amounts per ml of plasma used. Use the volume provided in the table for cfDNA purification from 10 ml
plasma.

(Optional) Proteinase K Treatment:
Proteinase K Treatment is required if samples were collected using Streck Cell-Free DNA BCT®
tubes. Omission of Proteinase K treatment may decrease yields by 50%. If blood was not collected using Streck Cell-Free DNA BCT® Tubes, proceed to Section B: Lysis/Binding.
Plasma volume

Proteinase K

20% SDS Solution

x (x = ml of plasma)0.015x

0.05x

0.5 ml7.5 µl

25 µl

1 ml

15 µl

50 µl

5 ml

75 µl

250 µl

10 ml

150 µl500 µl1.

Add the appropriate amount of plasma to an appropriately sized tube(s).

2.

Add 15 µl Proteinase K (20 mg/ml) for every 1 ml of plasma used.
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63.

Add 50 µl 20% SDS solution for every 1 ml of plasma used.

4.

Mix by inverting gently 5 times.

5.

Incubate at 60°C for 20 minutes.

6.

After incubation, place tube(s) on ice for 5 minutes to cool to room temperature.

7.

Once tube(s) have reached room temperature proceed directly into Section B: Lysis/Binding.

Section B: Lysis/Binding1.

Add the appropriate amount of plasma to the recommended sized tube(s).

2.

Add 1.25 ml of complete Lysis/Binding Buffer AM2 (with EtOH added) for every 1 ml plasma.

3.

Add 25 µl Magnetic Bead Solution for every 1 ml of plasma used.
Note:

4.

Mix bead solution prior to added to tubes. There should be no visible sedimentation at the bottom of the solution after mixing. Beads will settle quickly so it
is important to mix the magnetic bead solution after adding to each sample.
Failure to do so may result in inconsistent yields.

Vortex or shake tube(s) vigorously for 10 minutes at room temperature.
Note:

To obtain high yields, ensure that the plasma/buffer solution is mixing vigorously. A vortexing mixer with a tube-holder is recommended.

5.

Place tube(s) in a magnetic stand for 5 minutes, or until the solution clears.

6.

While keeping the tube(s) on the magnetic stand, remove and discard the supernatant. Be
careful not to disturb the magnetic particles.

7.

Keep tube(s) on the magnetic stand an additional minute. Then, remove and discard any
residual supernatant.

Section C: First Wash
1.

Remove the tube(s) from the magnetic stand and add 1 ml of complete Wash Buffer AM8(with EtOH added) to each Lysis/Binding tube.
Note:The same volume of buffer is used regardless of the tube size.

2.

For 2 ml and 15 ml tubes, resuspend the magnetic beads by vortexing for 10 seconds or by
pipetting up and down 6 times. For 50 ml tubes, resuspend beads by swirling tube or pipetting up and down 10 times.

3.

Transfer each magnetic bead suspension into a low retention 1.5 ml microcentrifuge tube.

4.

Place tube(s) in a magnetic stand for 10-30 seconds to allow the beads to aggregate to theside of the tube(s).

5.

Collect supernatant in pipette and use it to wash the original Lysis/Binding sample tube tocollect any remaining material. Transfer this solution back to the 1.5 ml microcentrifuge on
the magnetic stand and let sit for 10-30 seconds, or until solution is clear.
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Using a P1000 pipette, remove and discard as much supernatant as possible.

7.

Tap magnetic stand on bench 5 times to dislodge any remaining liquid. Using a P200
pipette, remove any remaining buffer.

8.

Remove tube(s) from the magnetic stand. Add 1 ml of complete Wash Buffer AM8 (withEtOH added) to each tube.

9.

Resuspend the magnetic beads by vortexing for 20 seconds or pipetting up and down 6times.

10. Quick spin the tubes in a microcentrifuge to collect the liquid to from the cap.
11.

Place tube(s) on a magnetic stand for 10-30 seconds.

12. Using a P1000 pipette, remove and discard as much supernatant as possible.
13. Tap magnetic stand on bench 5 times to dislodge any remaining liquid. Using a P200
pipette, remove any remaining buffer.

Section D: Second Wash
1.

Remove the tube(s) from the magnetic stand and add 1 ml fresh 80% EtOH.

2.

Resuspend the magnetic beads by vortexing for 20 seconds or by pipetting up and down 6times.

3.

Quick spin the tubes in a microcentrifuge to collect the liquid to from the cap.

4.

Place tube(s) in a magnetic stand for 10-30 seconds, or until solution is clear.

5.

Using a P1000 pipette, remove and discard as much supernatant as possible.

6.

Tap magnetic stand on bench 5 times to dislodge any remaining liquid. Using a P200pipette, remove any remaining ethanol.

7.

Remove tube(s) from the magnetic stand. Add 1 ml fresh 80% EtOH to each tube.

8.

Resuspend the magnetic beads by vortexing for 20 seconds or pieptting up and down 6times.

9.

Quick spin the tubes in a microcentrifuge to collect the liquid to from the cap.

10. Place tube(s) on a magnetic stand for 10-30 seconds.
11.

Using a P1000 pipette, remove and discard as much supernatant as possible.

12. Tap magnetic stand on bench 5 times to dislodge any remaining liquid. Using a P200pipette, remove any remaining ethanol.
13. Leave the tubes on the magnetic stand with the cap(s) open for 2 minutes. Then, tap magnetic stand on bench 5 times to dislodge any remaining liquid. Using a P20 pipette, remove
any remaining EtOH.
14. Allow magnetic particles to air dry for an additional 1-3 minutes.
Note:

Be careful not to over-dry. Over-drying may cause the beads to stick to the tube,
leading to a lower yield.

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8Section E: Elution
1.

Remove the tube(s) from the magnetic stand. and add the desired volume of Elution Buffer
AM6 to each tube. A minimum of 12.5 µl Elution Buffer AM6 per ml of plasma is recommended to elute DNA to ensure optimal yields.
Plasma volume

Elution Buffer AM6x (x = ml of plasma)0.0125x

0.5 ml6.25 µl

1 ml

12.5 µl

5 ml62.5 µl

10 ml

50 µl****When working with 10 ml of plasma per tube, the recommended amount of Elution Buffer AM6 differs from therecommended amounts per ml of plasma used. Use the volume provided in the table for cfDNA purification from 10 ml
plasma.

2.

Vortex tube(s) vigorously for 5 minutes.

3.

Quick spin the tubes in a microcentrifuge to collect the liquid to from the cap.

4.

Place tube(s) on a magnetic stand for 10-30 seconds.

5.

Transfer eluted cell-free DNA into a new microcentrifuge tube.

6.

Store DNA at -20°C, or proceed directly with downstream analysis.
Note:

Plasma will yield 1 - 100 ng of cell-free DNA per ml of plasma. Quantification
by absorbance measurement (e.g. Nanodrop) may not be sensitive enoughto accurately determine yields. Instead, we suggest using Qubit™ dsDNA HighSensitivity Assay.
Cell-free DNA is small in size (~170 bp). Therefore, PCR primers should be designed to produce amplicons of 150 bp or less. A higher number of amplification
cycles (40 cycles) may be needed in some cases.

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9Technical Services
If you need assistance at any time, please call or send an e-mail to Active Motif Technical Serviceat one of the locations listed below.

Active Motif North America
Toll free:Direct:Fax:
E-mail:Active Motif Europe
UK Free Phone:France Free Phone:
Germany Free Phone:Direct:Fax:
E-mail:877.222.9543
760.431.1263
760.431.1351
tech_service@activemotif.com
0800/169 31 47
0800/90 99 79
0800/181 99 10+32 (0)2 653 0001
+32 (0)2 653 0050
eurotech@activemotif.com

Active Motif Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
E-mail: japantech@activemotif.com
Active Motif China
Direct: (86)-21-20926090
Cell Phone: 18521362870
E-mail techchina@activemotif.com

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