Apr 02, 2025
Version 2
SPRI beads, variable PEG concentration V.2
- 1Max Planck Institute for Evolutionary Anthropology
- MPI EVA Ancient DNA Core Unit
Document Citation: Birgit Nickel, Anya Patova, Matthias Meyer 2025. SPRI beads, variable PEG concentration. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzj5n4lx1/v2Version created by Ancient DNA Core Unit
License: This is an open access document 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
Created: June 28, 2024
Last Modified: April 02, 2025
Document Integer ID: 125989
Keywords: SPRI beads
Funders Acknowledgements:
Max Planck Society
Grant ID: -
Abstract
This is a protocol for the preparation of beads suspension for the purification of nucleic acids using the Solid Phase Reversible Immobilization (SPRI) method (DeAngelis et al. 1995). Preparations of SPRI beads are used in various steps of sample preparation by the Ancient DNA Core Unit of the MPI-EVA as an alternative to commercially available products, such as AMPure XP beads (Beckman Coulter).
The size selectivity of SPRI beads depends primarily on the concentration of polyethylene glycol (PEG) in the reaction, which can be modulated by adjusting the PEG concentration in the bead suspension or by changing the volume ratio between SPRI bead suspension and sample. We here provide protocols for the preparation of bead suspensions containing 18%, 33% and 38% PEG-8000 (w/v) in three different volumes (50 ml, 100 ml and 250 ml). SPRI bead suspension containing 18% PEG performs similar to AMPure XP beads, which are often used in a 1.8x beads suspension/sample ratio. SPRI bead suspensions containing 33% or 38% PEG are useful if smaller volume ratios are desired, for example due to constraints in pipetting or reaction volumes. Instructions for DNA purification using these SPRI bead suspensions are provided in the protocols citing this document.
References
DeAngelis, M. M., Wang, D. G., Hawkins, T. L. (1995) Solid-phase reversible immobilization for the isolation of PCR products. Nucleic Acids Res. 23(22):4742-3. doi: 10.1093/nar/23.22.4742
Changes from v1
- A catalogue number and a supplier name that was missing in version 1 were added to the 125 ml square bottle in the consumables part of the materials section.
Note
SPRI bead suspension with 33% PEG also contains a slightly higher concentration of carboxyl coated beads to maximize the yield of DNA in the purification of indexing PCRs.
Materials
| Reagent/consumable | Supplier | Catalogue number | |
| Reagents | |||
| Sera-Mag SpeedBeads,15 ml, carboxylate-modified microparticles, 50 mg/ml | ThermoScientific | 6515-2105-050250 | |
| PEG 8000, Molecular Biology Grade | Promega | V3011 | |
| 5 M NaCl | Sigma Aldrich/Merck | S5150-1L | |
| 0.5 M EDTA, pH 8.0 | AppliChem | A4577,1000 | |
| 1 M Tris-HCl, pH 8.0 | AppliChem | A4892,1000 | |
| Tween-20 | Thermo Fisher Scientific | 11417160 | |
| TE buffer* | Self | ||
| UltraPure DNA/RNAse-free water | Sigma Aldrich/Merch | 1153332500 | |
| Consumables | |||
| Square media bottle 125 ml | Fisher Scientific | 10484481 | |
| Square media bottle 250 ml | VWR | 391-0629 | |
| Falcon tube, 50 ml, skirted | Greiner | GB 227270 | |
| 50 ml serological pipette | Corning BV | 357550 | |
* See document in the Appendix for preparation of TE buffer.
Equipment
- Serological pipette controller (e.g. battery powered pipetting aid ROTILABO, cat. no. TC16.1)
- Laboratory balance with 0.1g readability
- Magnetic rack (model depending on tube type)
Protocol for the preparation of SPRI bead suspension
1. Weigh PEG powder into a 50 ml Falcon tube, 125 ml or 250 ml square media bottle (depending on the volume of SPRI suspension desired) and add the following components as detailed in the table below. Fill up with water to the indicated volume using the graduation of the tube/bottle.
| Reagent | Mass/volume for 50 mL | Mass/volume for 100 mL | Mass/volume for 250 mL | Final concentration | |
| PEG 8000 | |||||
| - for 18% PEG | 9 g | 18 g | 45 g | 18% (w/v) | |
| - for 33% PEG | 17.5 g | 33 g | 82.5 g | 33% (w/v) | |
| - for 38% PEG | 19 g | 38 g | 95 g | 38% (w/v) | |
| 0.5 M EDTA, pH 8.0 | 100 µl | 200 µl | 500 µl | 1 mM | |
| 1 M Tris-HCl, pH 8.0 | 0.5 ml | 1 ml | 2.5 ml | 10 mM | |
| 5 M NaCl | 10 ml | 20 ml | 50 ml | 1 M | |
| Water | to ~45 ml | to ~95 ml | to ~240 ml |
2. Shake the tube or bottle until all PEG has dissolved.
3. Add the volume of Tween-20 indicated in the table below.
| Reagent | Volume for 50 mL | Volume for 100 mL | Volume for 250 mL | Final concentration | |
| Tween-20 | 25 µl | 50 µl | 125 µl | 0.05% (v/v) |
4. Mix by inverting or shaking the tube/bottle until the Tween-20 has completely dissolved.
5. Prepare Sera-Mag bead suspension:
5.1. Re-suspend stock solution of Sera-Mag beads by intense vortexing.
5.2. Transfer the volume of stock bead suspension indicated below to a 15 ml or 50 ml Falcon tube.
| Reagent | Volume for 50 mL | Volume for 100 mL | Volume for 250 mL | Final concentration | |
| Sera-Mag stock bead suspension (50 mg / ml) | |||||
| - for 18% PEG | 3 ml | 6 ml | 15 ml | 3 mg/ml | |
| - for 33% PEG | 3.55 ml | 7.1 ml | 17.75 ml | 3.55 mg/ml | |
| - for 38% PEG | 3 ml | 6 ml | 15 ml | 3 mg/ml |
5.3. Pellet the beads in a magnetic rack. Pipette off and discard supernatant.
5.4. Add 5 ml TE buffer and resuspend the beads by vortexing.
5.5. Pellet the beads in a magnetic rack. Pipette off and discard supernatant.
5.6. Repeat wash steps (5.4 and 5.5) once for a total of two washes.
5.7. Add 3 ml TE buffer and resuspend the beads by vortexing.
6. Add the complete volume of bead suspension to the tube/bottle from step 4. Fill up to 50, 100 or 250 ml with water using the graduation of the tube/bottle.
7. Mix bead suspension thoroughly by inverting the tube/bottle several times.
Note
[Labeling]
Label the bead suspension with "SPRI beads (XX% PEG)", date of production and the initials of the person who prepared the bead suspension.
Attention: Every single bottle prepared at the same day gets a new batch ID. Name the batches with Roman numerals (e.g. batch I, batch II, etc.)
8. Wrap the tube/bottle in aluminum foil (prevents light exposure) and store in the fridge for up to 1 year.
Note
[Note]
When preparing SPRI beads for the first time, use a DNA ladder to determine whether the size cutoff and DNA recovery are as expected (see Figure 1).
Fig. 1: Recovery of a DNA size marker (M: 50 bp ladder) using self-made SPRI bead suspension vs. a commercial product at different sample/bead suspension ratios.
Appendix
Document
NAME
TE bufferCREATED BY
Anna Schmidt