PCR Cleanup by SPRI (Solid-Phase Reversible Immobilisation) Beads
Purification of PCR products (amplicons) using paramagnetic SPRI beads. Removes primers, primer-dimers, dNTPs, salts, and polymerase enzymes from the amplicon, producing clean DNA suitable for Sanger sequencing and other downstream use. Works with commercial AMPure XP beads or DIY formulations from PEG + carboxyl-coated magnetic beads (~$0.05 per cleanup vs. $1–5 for commercial kits). Written for a novice audience.
Version History
Version 0.1.1 Latest
Effective: 2026-04-20Cross-reference fixes: agarose-gel refs 68→73, Sanger ref 71→75. Catalog fix (magnetic rack t#9) bundled. All `procedure N` references converted to Markdown hyperlinks pointing at https://librebiotech.org/?action=show&id=N — enables in-app click-through to referenced sibling procedures. Text content otherwise preserved.
Version 0.1.0 Viewing
Effective: 2026-04-20Initial release. SPRI bead-based cleanup is a field-standard technique developed from DeAngelis et al. 1995; now widely used with commercial (AMPure XP) and DIY formulations. Fresh original prose. Catalog gap: Magnetic rack equipment type not yet in LibreBiotech catalog — flag for addition.
Procedure Details
- Low hazard overall. SPRI beads are biologically inert; PEG is non-toxic; magnetic beads are safe handling.
- Ethanol. Flammable. Keep away from heat. Low toxicity; gloves standard.
- Magnetic rack handling. The neodymium magnets in commercial racks are powerful — keep away from pacemakers and metal-bearing items.
- Fresh ethanol matters. 80% ethanol diluted more than 24 hours ago absorbs water from humidity and becomes too dilute. Use fresh for best results.
- Pipetting hygiene. Filter tips throughout; work to avoid amplicon contamination of bench.
SPRI bead solution. Commercial (Beckman Coulter AMPure XP, ~$1/reaction) or DIY:
- 1.8 M NaCl + 10 mM Tris-HCl pH 8.0 + 1 mM EDTA + 20% PEG-8000 + 18 µL/mL carboxyl-coated magnetic beads (Sera-Mag SpeedBeads or equivalent).
- Mix thoroughly before every use — beads settle.
- Store 4°C; stable for 6+ months.
Fresh 80% ethanol. Prepare 80% v/v ethanol in water fresh each day. Older ethanol absorbs humidity and loses effectiveness.
TE elution buffer. 10 mM Tris-HCl pH 8.0 + 0.1 mM EDTA. Or use water for some downstream applications.
Magnetic rack. 96-well or PCR-strip format. Commercial options (Invitrogen DynaMag, Promega MagneSphere) or DIY neodymium magnet fixtures (see garage-genomics hardware repo).
Mental model: SPRI beads are paramagnetic beads coated with carboxyl groups. In the presence of PEG + NaCl, DNA binds to the beads reversibly. Binding is size-selective: higher bead:sample ratio retains smaller fragments; lower ratio only retains larger fragments. For PCR cleanup, 1.8× bead:sample ratio retains amplicons >150 bp while washing away primers and primer-dimers (<100 bp).
Bead ratio guide:
- 0.5×: retains only >1000 bp (removes most non-target)
- 1.0×: retains >300 bp
- 1.8×: retains >150 bp (standard PCR cleanup)
- 2.5×: retains all DNA (salt/enzyme cleanup only)
Critical timing: binding step is rate-limiting (5–10 min); washes are fast (30 s each).
- Per sample (batch-efficient on 8-tube strips or 96-well plates): 20 min hands-on.
- Scaling: 24 samples in parallel takes ~25 min with 8-channel pipette; >96 samples benefits from plate-based automation.
-
Microcentrifuge
Centrifugation
Specs: ≥13,000 × g (for brief spin-downs only)
Standard benchtop -
Micropipette
Liquid handling
Specs: P20, P200; filter tips
Standard set -
Vortex mixer
Mixing
Specs: Standard benchtop
For vortexing bead stock — NEVER for samples after binding
-
Buffer
Reagent
Qty: 45 µL per 25 µL PCR
1.8× ratio; commercial (AMPure XP) or DIY (PEG + NaCl + carboxyl-coated beads) -
Buffer
Reagent
Qty: 360 µL per cleanup (2 washes)
Prepare fresh daily; older ethanol loses effectiveness -
Buffer
Reagent
Qty: 20 µL per cleanup
10 mM Tris-HCl pH 8.0 + 0.1 mM EDTA; or water
Protocol Parameters Captured per-assay on each run; exported as ISA-Tab Parameter Value columns
| Name | Type | Required | Default | Unit | Description |
|---|---|---|---|---|---|
bead_ratio |
number | — |
1.8
|
— | Bead:sample volume ratio. 1.8× retains >150 bp (standard PCR cleanup); 1.0× retains >300 bp; 0.5× retains >1000 bp only. |
binding_time_min |
number | — |
10
|
minute (UO:0000031) | DNA binding time on beads. 10 min standard; shorter reduces recovery. |
magnet_separation_min |
number | — |
3
|
minute (UO:0000031) | Time for beads to settle on magnet. 2–5 min typical; larger tubes need longer. |
ethanol_wash_count |
number | — |
2
|
— | Number of 80% ethanol washes. 2 standard; 3 for salt-sensitive downstream applications. |
air_dry_time_min |
number | — |
3
|
minute (UO:0000031) | Air-drying time before elution. 2–5 min; too short = ethanol carryover; too long = overdried beads. |
elution_volume_ul |
number | — |
20
|
microliter (UO:0000101) | Elution volume. 20 µL standard; less concentrated higher, more diluted lower. |
elution_time_min |
number | — |
5
|
minute (UO:0000031) | Elution incubation time at room temperature. 5 min standard; 10 min for maximum recovery. |
Procedure Steps (Version 0.1.0)
Label fresh microcentrifuge tubes or strip wells for the cleanup. Place on the work surface alongside the magnetic rack.
Vortex the SPRI bead solution thoroughly for 30 seconds, then ensure it is homogeneous by inverting the bottle 5–6 times. Settled beads give inconsistent cleanups.
Add 1.8× volume of SPRI beads to your PCR product (e.g. 45 µL beads to 25 µL PCR). Mix by pipetting up and down 10 times — do NOT vortex; vortex shears beads and damages DNA.
Incubate at room temperature for 10 minutes. DNA binds to the beads during this step; the timing is critical and should not be rushed.
Place the tube/strip on the magnetic rack. Allow 2–5 minutes for the beads to collect against the magnet. The solution should become clear; the beads form a dark ring or pellet against the magnet side.
With the tube still on the magnet, carefully pipette off and discard the clear supernatant. Do NOT disturb the bead pellet. Leave a small volume (~5 µL) rather than risking bead aspiration.
Still on the magnet, add 180 µL of fresh 80% ethanol to the tube. Do NOT resuspend; just add on top of the beads.
Wait 30 seconds, then pipette off and discard the ethanol. The ethanol wash removes salts + PEG from the bound DNA.
Repeat the ethanol wash once more (add 180 µL, wait 30 s, remove).
Carefully pipette off any residual ethanol from the bottom of the tube with a P20 tip. Remove every visible drop.
Remove the tube from the magnet. Air-dry the bead pellet for 2–5 minutes. Watch for the pellet to transition from shiny/wet to matte/dry. Do NOT over-dry — cracked beads release DNA poorly.
Add 20 µL of TE buffer (or nuclease-free water for some downstream uses). Resuspend the beads by pipetting up and down 10 times until the solution is uniformly cloudy.
Incubate at room temperature for 5 minutes to allow DNA to elute off the beads.
Place the tube back on the magnetic rack. Allow 2 minutes for beads to collect.
Carefully transfer the clear eluate (~18 µL) to a new labelled tube. The DNA is in the supernatant; the beads stay behind on the magnet side.
Record the cleanup in LibreBiotech: Process record annotating bead ratio used, input sample ID, output sample ID. QC the cleaned product on an agarose gel (procedure 68) before downstream use.
Expected outcome. ~20 µL of clean DNA in TE buffer, concentration typically 60–80% of input. OD260/OD280 ratio 1.8–2.0 indicates clean DNA. Gel shows single clean band at expected size, no primer-dimer smear below 100 bp.
Typical recovery: 70–85% of input DNA for a 500 bp PCR amplicon with 1.8× bead ratio.
QC. Run 2 µL on an agarose gel (procedure 68) to confirm: single sharp band at expected size, no smear or secondary bands below. If Nanodrop available, check 260/280 ratio.
Downstream uses. Sanger sequencing submission (procedure 71); sub-cloning; enzymatic manipulation; quantitative downstream use.
Storage. 4°C for 1–2 weeks; −20°C for long term. Minimal freeze-thaw degradation.
Troubleshooting.
| Symptom | Likely cause | Fix |
|---|---|---|
| Very low DNA recovery | Elution with too-cold TE; insufficient elution time | Use room-temp TE; incubate 5 min at 37°C before magnet-separation |
| Primer-dimer still visible on gel | Bead ratio too high | Use 1.6× instead of 1.8× for stricter cutoff |
| Amplicon loss with fragment | Bead ratio too low for fragment size | Use 1.8× for <300 bp; 1.0× only for >1000 bp |
| Cloudy / beaded elution | Bead carryover into elution | Longer magnet settling time (2 min); pipette the clear supernatant only, leaving some behind |
| Ethanol smell in elution | Incomplete drying of bead pellet | Longer air-dry before elution (verify no liquid visible); but don't over-dry (DNA hard to elute from overdried beads) |
References
- DeAngelis MM, Wang DG, Hawkins TL (1995). Solid-phase reversible immobilization for the isolation of PCR products. Nucleic Acids Res 23(22):4742–3. (Original SPRI method paper). DOI paper
- Rohland N, Reich D (2012). Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res 22(5):939–46. (DIY SPRI formulation). DOI paper