AMPure XP / SPRI Bead Size Selection

Allow AMPure XP beads to equilibrate to room temperature for at least 30 minutes. Vortex thoroughly to resuspend beads before use.

Determine the bead ratio for the desired size cutoff. Common ratios:

For ONT PCR-cDNA size selection at 1.5 kb, use 0.4×.

Prepare the sample:

• Adjust DNA concentration to approximately 60 ng/µL in TE buffer (pH 8.0)
• Use a final sample volume of 50 µL (dilute with TE if necessary)
• Transfer to a 1.5 mL DNA LoBind tube

Add the calculated volume of resuspended AMPure XP beads to the sample (e.g. for 0.4× ratio with 50 µL sample, add 20 µL beads). Mix thoroughly by flicking the tube or pipetting up and down 10 times. Do not vortex.

Incubate at room temperature for 10 minutes to allow DNA binding. Flick the tube to resuspend the beads halfway through the incubation.

Place the tube on a magnetic rack. Wait until the solution clears completely (approximately 2–5 minutes).

Collect the supernatant — this contains the large fragments that did NOT bind to the beads (size-selected fraction). Transfer the supernatant carefully to a new tube. Discard the pellet.

Note: This is the opposite of a cleanup — in size selection, the supernatant contains the desired large fragments.

Add a second round of beads to the supernatant at a high ratio (e.g. 1× volume) to capture the size-selected fragments. Mix by flicking and incubate at room temperature for 10 minutes.

Place on the magnetic rack until the solution clears. Remove and discard the supernatant. The size-selected DNA is now bound to the beads.

Wash the bead pellet twice with 200 µL freshly prepared 70% ethanol:

• Add ethanol, wait 30 seconds
• Remove ethanol completely
• Repeat
• Briefly spin down and remove residual ethanol with a fine tip
• Air-dry the pellet for approximately 30 seconds (do not over-dry)

Elute the DNA by resuspending the bead pellet in 12 µL nuclease-free water or TE buffer. Incubate at room temperature for 5 minutes, then place on the magnetic rack and collect the eluate (~11 µL recovered).

Quantify the eluted library using Qubit dsDNA HS assay. Optionally verify the size distribution using Fragment Analyzer or TapeStation to confirm depletion of short fragments below the cutoff.