Mar 17, 2026

xGen cfDNA & FFPE DNA Library Prep v2 MC

This  protocol  is a draft, published without a DOI.
  • 1Washington University
  • HVP Pregnancy Postpartum
Kristine Wylie
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Protocol Citation: Kristine Wylie, Jane Schrimpf, Madison Eschbach, Hunter Olson 2026. xGen cfDNA & FFPE DNA Library Prep v2 MC. protocols.io https://dx.doi.org/
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 16, 2026
Last Modified: March 17, 2026
Protocol  Integer ID: 313349
Keywords: ffpe dna library prep v2 mc kit, ffpe dna library prep v2 mc protocol, idt xgen cfdna, xgen cfdna
Funders Acknowledgements:
NIH NCCIH
Grant ID: U01 AT012970
Abstract
Protocol was adapted from the IDT xGen cfDNA & FFPE DNA Library Prep v2 MC Kit.
Materials
- AMPure beads
- PEG/NaCl
- 80% ethanol in molecular biology grade water
- End Repair Buffer
- End Repair Enzyme
- Ligation 1 Buffer
- Ligation 1 Adapter
- Ligation 1 Enzyme
- Ligation 2 Buffer
- Ligation 2 Adapter
- Ligation 2 Enzyme A
- Ligation 2 Enzyme B
- xGen UDI Primer Pairs
- xGen 2X HiFi PCR Mix
- EB
Safety warnings
IMPORTANT: Before starting protocol, ensure that the AMPure beads and PEG/NaCl are at room temperature. Also, prepare fresh 80% ethanol in molecular biology grade water.

Safe Stopping Point: samples can remain at 4°C for no more than 2 hours.
Safe Stopping Point: samples can be stored at -20°C overnight.
Before start
Before starting protocol, ensure that the AMPure beads and PEG/NaCl are at room temperature. Also, prepare fresh 80% ethanol in molecular biology grade water.
End Repair
Add 50 µL of each sample to 8-tube strip of PCR tubes.
Make up End Repair Master Mix and keep on ice:


Add 9 µL of End Repair Master Mix to each tube and mix by pipetting up and down 10 times.
Close tube and place in thermocycler and run End Repair program with thermocycler lid OFF.


While the program runs, make up Ligation 1 Master mix and place on ice:


When the program reaches 4°C, proceed immediately to post-end repair cleanup.
End Repair Cleanup
Add 47.2 µL AMPure beads (0.8X volume) to each sample and pipet up and down to mix.
Incubate tubes at room temperature for 10 min.
Place tubes on the magnet and wait for liquid to clear (at least 2 min).
Remove and discard supernatant.
Quick spin and remove and discard any trace amount of supernatant that remains.
Keeping tubes on the magnet, add 160 µL of 80% EtOH and incubate for 30 sec.
Remove and discard supernatant.
Quick spin and remove and discard any trace amount of supernatant that remains. DO NOT DRY.
Proceed immediately to Ligation 1.
Ligation 1
Remove the plate from the magnet and resuspend beads in 30 µL Ligation 1 Master Mix. Pipette to mix a minimum of 10 times.
Place tubes in thermocycler and run Ligation 1 program with thermocycler lid ON.


Safe Stopping Point: samples can remain at 4°C for no more than 2 hours.
Proceed to Ligation 2.
Ligation 2
Prepare Ligation 2 Master Mix and keep on ice:


Add 10 µL Ligation 2 Master Mix to each tube and mix by pipetting up and down 10 times.
Place tubes in thermocycler and run Ligation 2 program with thermocycler lid ON.


When the program reaches 4°C, proceed immediately to post-ligation 2 cleanup.
Ligation 2 Cleanup
Add 32 µL PEG/NaCl (0.8X volume) to each tube and pipette 10 times to mix.
Incubate tubes at room temperature for 10 min.
Place tubes on the magnet and wait for liquid to clear (at least 2 min).
Remove and discard supernatant.
Quick spin and remove and discard any trace amount of supernatant that remains.
Keeping tubes on the magnet, add 160 µL of 80% EtOH and incubate for 30 sec.
Remove and discard supernatant.
Quick spin and remove and discard any trace amount of supernatant that remains. DO NOT DRY.
Remove the tubes from the magnet and resuspend beads in 20 µL EB.
Incubate the tubes at room temperature for 5 min.
Place the tubes on the magnet and wait 1-2 min for the liquid to clear.
Carefully transfer 20 µL of the supernatant containing the eluted DNA into a fresh tube.
Proceed to PCR amplification or pause here.
Safe Stopping Point: sample can be stored at -20°C overnight.
PCR Amplification
Add 5 µL of xGen UDI Primer Pairs to each tube.
Add 25 µL of xGen 2X HiFi PCR Mix to each tube and pipette up and down 10 times to mix.
Place tubes in thermocycler and run PCR program with thermocycler lid ON.


After the program is complete, proceed to post-PCR cleanup.
PCR Cleanup
Add 40 µL AMPure beads (0.8X volume) to each tube and pipette up and down 10 times to mix.
Incubate the tubes at room temperature for 5 min.
Place tubes on the magnet and wait for liquid to clear (at least 2 min).
Remove and discard supernatant.
Quick spin and remove and discard any trace amount of supernatant that remains.
Keeping tubes on the magnet, add 160 µL of 80% EtOH and incubate for 30 sec.
Remove and discard supernatant.
Quick spin and remove and discard any trace amount of supernatant that remains. DO NOT DRY.
Remove the tubes from the magnet and resuspend beads in 31 µL of EB.
Incubate the tubes at room temperature for 5 min.
Place the tubes on the magnet and wait 1-2 min for the liquid to clear.
Carefully transfer 30 µL of the supernatant containing the eluted DNA into a fresh tube and store at -80°C.
Quality Control
Measure DNA concentration using Qubit HS DNA assay.
Run libraries on an Agilent DNA 12000 bioanalyzer chip to determine final library size distribution and confirm minimal primer or adapter dimer carryover.