Home › Molecular Biology › How to Isolate Cell-Free DNA from Plasma | cfDNA Purification
Steps
  1. 1 Prepare plasma by centrifugation 00:27
  2. 2 Mix lysis buffer with magnetic beads 00:58
  3. 3 Bind DNA to magnetic beads 01:23
  4. 4 Wash beads with buffer solutions 01:41
  5. 5 Elute purified cell-free DNA 03:33
  6. 6 Collect and store purified DNA 04:02
  7. 7 Automate isolation with magnetic processor 04:31
  8. 8 Transfer and store automated results 05:05
Molecular Biology Thermo Fisher Scientific

How to Isolate Cell-Free DNA from Plasma | cfDNA Purification

Protocol
Difficulty
intermediate

Steps

1
Prepare plasma by centrifugation

Centrifuge the blood collection tube at 2,000 G for 10 minutes and transfer plasma to a fresh tube. Centrifuge again at 16,000 G for 10 minutes and transfer the cleared plasma to another fresh tube.

▶ 00:27
2
Mix lysis buffer with magnetic beads

Add 2.5 mL of lysis binding solution and 30 microliters of M1 silane beads to a fresh conical tube and mix by vortexing.

▶ 00:58
3
Bind DNA to magnetic beads

Add 2 mL of plasma sample, mix by vortexing, and incubate for 10 minutes while shaking or rotating to allow DNA binding to the beads.

▶ 01:23
4
Wash beads with buffer solutions

Pellet magnetic beads using a magnetic stand and discard supernatant. Perform sequential wash steps with wash solution and 80% ethanol, pelleting beads between each wash and removing supernatant carefully.

▶ 01:41
5
Elute purified cell-free DNA

Add 50 microliters of DNA elution solution to the dried beads, vortex for 5 minutes, and briefly centrifuge. Place tube on magnetic stand for 2 minutes until solution clears and beads are pelleted.

▶ 03:33
6
Collect and store purified DNA

Transfer the purified cell-free DNA solution to a non-stick microcentrifuge tube and store at minus 20 degrees Celsius for long-term storage, or use immediately.

▶ 04:02
7
Automate isolation with magnetic processor

Prepare processing plates according to the product manual, add plasma samples to rows A and B, and load into the Kingfisher Flex or Duo instrument. Select the appropriate program and start the run.

▶ 04:31
8
Transfer and store automated results

Remove processing plates at the end of the run and transfer eluted cell-free DNA to an elution plate. Store at minus 20 degrees Celsius for long-term storage or use immediately.

▶ 05:05

🚨 Failure Case Library (8) + Submit your own case

severe
Low cfDNA Yield Due to Improper Reagent Handling
Cell-free DNA extraction yields are lower than expected. This may manifest as insufficient DNA concentration measured by fluorometry or qPCR, preventing downstream analysis.
💡 4 · ✓ 4
severe
Low cfDNA Recovery from Inadequate Bead Dispensing
DNA yield is consistently lower than expected across multiple extractions. The binding capacity appears reduced, with proportionally less DNA recovered relative to input sample volume.
💡 4 · ✓ 4
severe
Poor cfDNA Binding from Inadequate Incubation
Cell-free DNA yield is suboptimal despite proper bead addition and reagent preparation. A significant portion of DNA may remain unbound in the supernatant during the binding step.
💡 4 · ✓ 4
severe
Yield or Quality Issues from Incorrect Bead Drying
DNA yield is lower than expected, or downstream applications show inhibition. Under-dried beads may carry over ethanol or contaminants, while over-dried beads result in poor DNA release during elution.
💡 4 · ✓ 4
severe
Low cfDNA Recovery from Incomplete Elution
Final DNA concentration is lower than expected despite successful binding and washing. A significant portion of DNA remains bound to the beads after the elution step.
💡 4 · ✓ 4
moderate
DNA Loss from Bead Removal During Supernatant Transfer
Variable or reduced cfDNA yields across samples. Beads may be visible in wash supernatants or final eluate, or yield decreases progressively through the protocol.
💡 4 · ✓ 5
moderate
Inherently Low cfDNA Yield from Sample Limitations
Consistently low cfDNA yields despite optimized extraction protocol. This pattern may occur across specific sample types or patient cohorts, reflecting biological rather than technical causes.
💡 4 · ✓ 5
moderate
Magnetic Bead Contamination in Final cfDNA Eluate
Visible brown particles or turbidity in the final DNA eluate. This may cause downstream assay interference, optical measurement errors, or sequencing library preparation issues.
💡 4 · ✓ 5
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