Troubleshooting Guide for Monarch^® Mag Cell-free DNA (cfDNA) Extraction Kit (NEB #T4070)

Lower cfDNA yield that expected

Incorrect reagent preparation or storage.

Ensure that all components are stored at the appropriate conditions for optimal performance. Monarch Mag Beads M2 are stored at 4̊ C, but should be allowed to warm to room temperature before use. Check the protocol to confirm that buffers have been properly prepared and that the correct volumes are used. Verify that the appropriate buffer is added at each step.

Insufficient beads added

Ensure that the Monarch Mag Beads M2 solution is thoroughly mixed before addition to the sample. Because the beads settle quickly, inadequate mixing may result in dispensing fewer beads than required, reducing binding capacity and lowering DNA yield. Use low-retention pipette tips to avoid potential loss of beads on the tip walls.

Insufficient binding

It is imperative to allow the sample to mix with the bead-binding mix for 10 minutes to create optimal conditions for cell-free DNA to bind to the beads.

Beads not optimally dried

Beads should be dried for 5 minutes while the tubes remain on the magnetic rack with the tube caps open. Under-dried beads can lead to contaminant carryover and over-dried beads may lead to lower yield. Beads that are over-dried appear matte and light brown, often with visible surface cracks. Optimally dried beads appear shiny and uniformly dark.

Incomplete elution

Thorough mixing of the beads with the Nuclease-free Water is essential for eluting DNA off the beads. Pipette mix well to ensure that all beads are resuspended, then incubate for 5 minutes on a thermal mixer/shaker.

Beads lost during prep

Use a strong magnet to ensure effective bead separation, and carefully remove the supernatant without disturbing the bead pellet, as any beads removed with the supernatant may result in DNA loss.

Sample contains low levels of cfDNA

Sample collection processes and biological variability can result in inherently low cfDNA loads in the starting sample. Increasing sample input volume can help.

Magnetic bead carryover

Beads not magnetized sufficiently

During magnetic separation steps, ensure that the supernatant is clear and free of beads before removing it. Leave the tubes on the magnet for a minimum of 2 minutes at all magnetic separation steps.

Improper pipette handling

When transferring the final elution to a new tube, be careful not to disturb the bead pellet.