Sep 10, 2025

Public workspaceClean up of Index PCRs using SPRI Beads V.2

DOI
https://dx.doi.org/10.17504/protocols.io.36wgqqkdxgk5/v2
  • Michael V. Westbury1,
  • Alba Rey-Iglesia1,
  • Vanssy Li1,
  • Deon de Jager1,
  • Eline Lorenzen1
  • 1University of Copenhagen
  • Lorenzen Lab
Vanssy Li
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DOI: https://dx.doi.org/10.17504/protocols.io.36wgqqkdxgk5/v2
Protocol Citation: Michael V. Westbury, Alba Rey-Iglesia, Vanssy Li, Deon de Jager, Eline Lorenzen 2025. Clean up of Index PCRs using SPRI Beads. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqqkdxgk5/v2Version created by Deon de Jager
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: September 09, 2025
Last Modified: September 10, 2025
Protocol Integer ID: 226817
Keywords: cleanup of index pcr product, clean up of index pcr, using spri bead, index pcr product, spri bead, index pcr, dna libraries with concentration measurement, spri beads this protocol, purified dna library, dna elution, pcr, dna ratio, bead
Abstract
This protocol describes the cleanup of index PCR products using SPRI bead-based size selection at a 1.4:1 bead-to-DNA ratio. The process includes bead binding, ethanol washes, DNA elution, and quantification using Qubit or other methods. The expected result is purified DNA libraries with concentration measurements for downstream analysis.
Protocol materials
ReagentSpeedBeads magnetic carboxylate modified particlesMerck MilliporeSigma (Sigma-Aldrich)Catalog #GE45152105050250
ReagentEB bufferQiagenCatalog #19086
Reagent1.5mL DNA LoBind tubesEppendorfCatalog #0030108051
Troubleshooting
Before start
This protocol is processed in the modern lab. Clean all surfaces with 5% bleach followed by 70% ethanol and all equipment with 70% ethanol before and after use.

Reagents and Materials
Before starting, make sure you have the following materials ready:
  • ReagentSpeedBeads magnetic carboxylate modified particlesMerck MilliporeSigma (Sigma-Aldrich)Catalog #GE45152105050250 (aka "SPRI beads"). Equilibrate to room temperature and vortex thoroughly before use. Can also use other types/brands of SPRI beads (e.g. AMPure XP Beads for DNA Cleanup).
  • 80% ethanol (EtOH)
  • ReagentEB bufferQiagenCatalog #19086
  • Magnetic rack
  • Reagent1.5mL DNA LoBind tubesEppendorfCatalog #0030108051


Prepare fresh 80% EtOH in an appropriately sized tube (e.g. 15 mL or 50 mL falcon tube). You need 400µL per sample, and you have n samples.
100% EtOH (µL)ddH2O (µL)per sample (µL)
320 80400
n+1 * 320n+1 * 80n+1 * 400

SPRI Bead Cleanup
5m

Note



Add 70 µL SPRI beads to a 1.5 mL tube.
Add 50 µL index PCR product to the same tube. Mix them well by pipetting. Spin down very briefly in a microcentrifuge to collect beads - check that you did not pellet the beads at the bottom of the tube. If you did, resuspend by pipetting.
Incubate at room temperature for at least 5 minutes.

THIS IS A CRUCIAL STEP. This incubation allows the DNA to bind to the beads, so it’s essential to avoid moving them and have enough time for the DNA to bind to the beads.
5m
Incubation
Place tubes on magnetic rack until solution clears (~2-5 min).
Remove supernatant using a pipette while the tubes are still in the magnetic rack (~110 µL).
Ethanol Wash
Add 200 µL 80% EtOH while the tubes remain on the magnetic rack.
Remove ethanol carefully with a pipette. Be careful not to remove any beads.
Add another 200 µL 80% EtOH.
Remove ethanol again.
Close lids loosely.
Drying
Open lids, air-dry beads for ~2 minutes. (Use timer)
  • Important: Do not over-dry, as this will destroy the DNA.
  • Use the small pipette to remove residual EtOH during these 2 minutes. Work quickly!
After drying for 2 min, close the lids.
Elution
Leave the tubes still in the magnetic rack. Add 25–32 µL EB buffer (typically 27 µL) to elute the DNA.
Remove the tubes from the magnetic rack. Flick/vortex to resuspend.
Incubate 10 minutes at room temperature or 37°C. This is where the DNA is released from the beads into the elution buffer.
While waiting for the 10-minute incubation, label new LoBind tubes - these are your final tubes.
Place the tubes back in the magnetic rack and wait until the beads pellet (the DNA is now in solution).
Transfer 25–32 µL (typically 25-26 µL) of supernatant to a clean, labeled LoBind tube. Be careful not to transfer any beads, as these will have negative effects on downstream applications, such as sequencing!
QC (Qubit or Fragment Analyser, or Bioanalyser etc.)
Following is the Qubit protocol to get the concentration of the libraries.
Typically, we would also run the indexed, cleaned libraries on a Fragment Analyzer (FA) or Bioanalyzer (BA) (TapeStation is also fine) to get the fragment size of the libraries, which is important when pooling libraries for sequencing. (Note that the FA, BA, and TapeStation have maximum concentration limits, so check their documentation and dilute your samples as needed, based on their Qubit concentrations.)
Prepare Qubit working solution (WS):
  • Remove the Qubit standards from the fridge now to equilibrate to room temperature before use.

Assuming you have n samples, and 2 standards.
AHS buffer (µL)Qubit HS Dye (µL)per sample (µL)
One sample1991200
n samples199 * (n+2)1 * (n+2)200 * (n+2)

For samples:
Add 199 µL WS to each Qubit tube + 1 µL library. (You can use up to 5 µL of sample - just adjust the volume of WS accordingly. But even for aDNA libraries, we use only 1 µL.)
For standards:
Add 190 µL WS + 10 µL standard 1.
Add 190 µL WS + 10 µL standard 2.
Measure on Qubit:
Follow Qubit instructions to make measurements.
Set sample volume to 1 µL. Set output units to ng/µL.