- Ideal for extracting DNA from up to 5 mL human plasma and serum for cell-free DNA sequencing and other downstream applications
- Highly automated, mag-bead based format with robust yields
- Validated with the chemagic™ 360 or chemagic™ MSMI instruments
- Fast, 15 minutes hands-on-time and 1.25 hour turnaround for 24 samples
- Approximately 128 samples extractions possible per day
The NextPrep-Mag™ cfDNA Automated Isolation Kit is a complete solution for automated cell-free DNA extraction from human blood plasma or serum using the chemagic™ 360, chemagic™ Prime™ or chemagic™ MSMI instruments from PerkinElmer®.
The challenges of extraction of cfDNA from plasma or serum are several, but are rooted in the fact that the circulating DNA is highly fragmented and present at low concentrations, especially in healthy individuals. The NextPrep-Mag™ cfDNA Automated Isolation Kit was developed to facilitate the workflow demands for high throughput labs while delivering optimal results.
The NextPrep-Mag™ cfDNA Automated Isolation Kit allows for the extraction of cell-free DNA from human blood plasma or serum using the chemagic™ 360, chemagic™ prime or chemagic™ MSMI instruments. The kit is all-inclusive and contains ready-to-use reagents and disposable plastics for 240 automated cfDNA isolations from up to 5 mL of starting plasma/serum. The cfDNA recovered can be used in a wide variety of downstream applications including next-gen sequencing and PCR analysis.
The cfDNA isolated with the NextPrep-Mag™ cfDNA kits have been verified to be compatible with both manual and automated NGS library construction on the Sciclone® G3 NGS robot using the NEXTflex® Cell Free DNA-Seq Library Prep kit for Illumina® sequencing.
For research use only. Not for use in diagnostic procedures.
Automated cfDNA Isolation Protocol
The automated process allows for a significant decrease in total turnaround time and hands on time compared to current manual and partially automated solutions on-market.
NextPrep-Mag™ cfDNA Automated Isolation Kit Design Highlights
The kit offers a number of key advantages over other commercially available cfDNA extraction kits, including speed of cfDNA extraction while delivering robust yields and high concentration NGS libraries.
NextPrep-Mag™ cfDNA Automated Isolation Kit Performance
The kit generated robust cfDNA recovery from both plasma and serum on the chemagic™ 360 and chemagic™ MSMI platforms as analyzed by the Agilent® BioAnalyzer® 2100 Instrument and Qubit.
Figure 1: Performance on the chemagic™ 360 Instrument. cfDNA was extracted from plasma (PL) or serum (SER) from 5 donors. (A) Yields were quantified using the Agilent® Bioanalyzer® Instrument (for mononucleosome peak) and Qubit (for total DNA yield). (B) Overlay traces of triplicate 4 mL extractions.
Figure 2: The NextPrep-Mag™ cfDNA Automated Isolation Kit is robust on the chemagic™ MSMI instrument. Blood from different donors was collected in either (A) SST tubes or (B) Streck BCT tubes and extracted for serum. Serum was pooled from each collection method and 5 mLs were extracted using the automated protocol.
Downstream sequencing application of cfDNA extracted using the NextPrep-Mag™ chemistry
cfDNA extracted from Horizon Discovery’s cfDNA Standards using the NextPrep-Mag™ chemistry cfDNA generated high quality sequencing libraries and excellent concordance with expected mutation frequencies down to 0.1% using a commercially available amplicon kit.
Figure 3: Sequencing results from cfDNA extracted using NEXTPrep-Mag™ chemistry shows good concordance with expected mutation frequencies (A, B).
The NextPrep-Mag™ cfDNA Automated Isolation Kit contains sufficient reagents for 240 isolations of 4 – 5 mL plasma or serum along with the accompanying disposable plastics for the chemagic™ 360 automation. The kit components are stable for at least 12 months from time of receipt, when properly stored.
cfDNA Lysis/Binding Solution
cfDNA Proteinase K
cfDNA Magnetic Beads
cfDNA Wash 1 Solution
cfDNA Wash 2 Solution (user adds 135 mL ethanol)
cfDNA Elution Solution (10 mM Tris/0.1 mM EDTA, pH 8)
chemagic™ 360 PLATFORM PLASTICS
13 mL tubes
4 mL elution tubes
Required Materials Not Provided
Distilled deionized water used to dissolve Proteinase K
5 mL disposable pipets
chemagic™ 360 Platform Requirements
3 of the Double bottom rack adapters (CMG-13005340 ), not included with instrument purchase
1 of the 24-Rod head set (includes the 24-Deepwell plate adapter) (CMG-376) if not part of original instrument setup
1 set of the 1 L bottles (6 included) with adapter lids (CMG-588)
Do we need special equipment to process large volumes of plasma?
No, the same kind of clinical centrifuges are used. For Input volumes less than or above 5 mL, slight modifications to the protocol may be required. Please inquire directly to Bioo.NGS@PerkinElmer.com.
What is the size distribution of cell free DNA recovered from human plasma?
The cell free DNA isolated from plasma of healthy donors, typically runs as a single small “hump” of ~ 150 bp when analyzed directly on an Agilent 2100 high sensitivity DNA chip. When the cell free DNA is converted into a library using the ligation-based format in the NEXTflex® Cell Free DNA-Seq Library Prep Kit, a greater range of sizes of library products is observed, from ~ 300 bp – 5,000 bp. The 5’ and 3’ adapters used to make the library add a total of ~150 bp. This indicates that cell free DNA fragments present at concentrations too low to be visualized directly are detectable after the amplification step used to create cell free DNA libraries.
Is it necessary to add carrier nucleic acid or glycogen when processing plasma to recover cell free DNA?
No. Addition of nucleic acid carrier compromises quantification and use of the cell free DNA as input for NGS libraries.
How many PCR cycles are needed to make cell free DNA libraries?
We typically use 9 – 12 PCR cycles for making cell free DNA libraries.