Jul 03, 2025
18% SPRI reagent
- Nadin Rohland1
- 1Harvard Medical School
Protocol Citation: Nadin Rohland 2025. 18% SPRI reagent. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l21mr4g1y/v1
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: July 02, 2025
Last Modified: July 03, 2025
Protocol Integer ID: 221619
Keywords: ampure xp bead, reagent, brew reagent
Abstract
This protocol explains how to prepare 18% SPRI beads that can be used instead of AMPure XP beads.
Note that as with any home-brew reagent, batches can show different behavior and should be tested (with a DNA ladder) prior to use.
Guidelines
This reagent, if stored in the refrigerator and covered from light, can last for up to 2 years (and potentially longer) in our experience.
Keep the stock at 4C in the dark and make smaller working aliquots after the beads are well suspended.
Materials
Magnetic racks for different tubes/plates:
- 50ml magnetic separation rack (e.g. NEB: S1507S or V&P Scientific: VP 772FB-2)
- 1.5/2.0ml magnetic separation rack (e.g. DynaMag-2 Magnet; ThermoFisher Scientific: 12321D)
- 96-well plate separation rack (ring magnet works well for automation, e.g. 96S Super Magne;, Alpaqua Engineering: A001322)
- 96-well plate separation rack (bar magnet or other magnets for 96 well plates work well for manual multichannel pipetting, e.g. Invitrogen: 12027 or Maxi Scientific: 4E-MRK100296)
1 Liter bottle, e.g. Nalgene Square PET Media Bottles with Closure, ThermoFisher Scientific: 342040-1000
| item | vendor | part number | |
| Sera-Mag Speedbead carboxylate-modified [E3] magnetic particles | Cytiva | 65152105050250 (15ml) | |
| PEG-8000 | Millipore- Sigma | 89510 | |
| 5M NaCl | Millipore- Sigma | S5150 | |
| 1M Tris-HCl | Invitrogen | 15568025 | |
| 0.5M EDTA | Millipore- Sigma | 03690 | |
| water | Millipore- Sigma | W4502 | |
| optional: GeneRuler Ultra Low Range DNA ladder | Thermo Scientific | FERSM1211 |
Prepare TE for bead washing and resuspension
| reagent | volume | final concentration | |
| 1M Tris-HCl | 10 | 10 mM | |
| 0.5M EDTA | 2 | 1 mM | |
| water | 988 | ||
| TOTAL | 1000 |
Troubleshooting
Safety warnings
Mix Sera-Mag Speedbeads well before washing and SPRI reagent before using or aliquoting.
Before start
This protocol is for 750ml 18% SPRI reagent (using a single bottle of 15ml of the Sera-Mag Speedbeads).
If you make less, proportionally downscale.
Wash beads
For a total of 750ml 18% SPRI reagent, use the entire 15ml bottle of Sera-Mag Speed beads carboxylate-modified and vortex vigorously to remove thick aggregates of beads; transfer into a 50ml Falcon tube.
Add 20 ml TE to rinse the storage bead bottle, mix and transfer to the Falcon tube; mix well by vortexing, then briefly centrifuge tube to collect liquid from inside the lid (e.g. by swinging your arm or short low centrifugation)
Place tube in magnet to collect beads on tube wall, this takes about 10 minutes or until liquid is clear
Remove supernatant with serological pipet and add between 20 and 35 ml TE, mix well to wash beads more by vortexing.
Repeat steps 3 and 4 for a total of 2 washes, and resuspend washed beads in 15ml TE.
preparation of 18% SPRI reagent
Weigh out 135 g of PEG-8000 into a 1 Liter bottle.
To the PEG-8000 add below reagents
| reagent | volume in ml | final concentration | |
| 5M NaCl | 150 | 1 M | |
| 1M Tris-HCl | 7.5 | 10 mM | |
| 0.5M EDTA | 1.5 | 1 | |
| washed beads from Step 5 | 15 | 0.1% solid particles |
and fill up with water to 750 ml.
Mix well and let stand until all PEG particles are dissolved; intermittent shaking is helpful
Label with date and store in a dark box (or cover with aluminum foil) in the refrigerator.
This reagent is comparable to AmPure XP beads and can be used as recommended for the original product.
(The recommended volume is 1.8x for standard PCR cleanup to retain dsDNA > 100bp and clean away shorter primers and adapters etc)
Optional: preparing of 38% SPRI reagent
If a SPRI reagent is needed that allows to retain shorter fragments and to work in smaller volumes (e.g. to fit in a specific plate for automation), prepare a 38% SPRI reagent by following the above, but use 285 g of PEG-8000.
The 38% SPRI reagent can be used in equal volume to the cleanup PCR reactions from e.g. ancient DNA library preparations.
Recommended: testing of new reagent batch
Use 5 μl (2.5 μg total) of GeneRuler Ultra Low Range DNA ladder
Add 95 μl water
add 180 μl 18%SPRI (1.8x as recommended for AMPure XP)
(or 100 μl 38% SPRI)
mix by vortexing or pipetting
incubate 5 min at room temperature
spin and place on appropriate magnet for 4 minutes or until liquid is clear (it takes longer for the more viscous 38%SPRI)
wash beads while tubes remain on magnet with fresh 80% ethanol twice
spin, put back on magnet and remove remaining ethanol with pipet
air dry for 1min at room temperature
elute in 20 μl TE by resuspending the beads by pipetting
spin, place on magnet and let separate for 1min
collect elution into new tube
visualize on BioAnalyzer or TapeStation together with diluted original ladder of comparable starting amount
18% SPRI: the 100 bp band should be less strong and the shorter fragments should be gone
38% SPRI: the 50 bp should be as strong as before the cleanup and 35 bp should be more faint, 25 bp band should be gone.
Protocol references
DeAngelis MM, Wang DG, Hawkins TL. Solid-phase reversible immobilization for the isolation of PCR products. Nucleic Acids Res. 1995 Nov 25;23(22):4742-3. doi: 10.1093/nar/23.22.4742. PMID: 8524672; PMCID: PMC307455.
Rohland N, Reich D. Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res. 2012 May;22(5):939-46. doi: 10.1101/gr.128124.111. Epub 2012 Jan 20. PMID: 22267522; PMCID: PMC3337438.
Acknowledgements
I'd like to thank Matthias Meyer and his team at MPI-EVA for developing the 38% SPRI version.