# Protocol for AMPure XP beads substitute

##### *version 1.0: 2023-06-14*

### Overview

The objective of this protocol is to create a substitute for AMPure XP beads with equal effectiveness in comparison to the commercial product, but more cost-effective ($19/mL versus $0.46/mL).

This protocol is derived from the referenced protocol created by Nadin Rohland and David Reich - subsequently adapted by Brant Faircloth. If you use this protocol, **please** ensure that you cite their work:

*Citation:* land N, Reich D. [Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture](https://genome.cshlp.org/content/22/5/939). ***Genome Research 22***: 939-946.

---
**Materials**

Below is a list of stock solutions that can be purchased pre-mixed and sterilized. This is in an attempt to minimize variation to the degree possible. You can certainly prepare your own stock solutions at appropriate pH.

• Sera-mag beads (Fisher # 09-981-123; GE # 65152105050250)
• PEG-8000 (Amresco 0159)
• 0.5M EDTA, pH 8.0 (Amresco E177)
• 1.0M Tris, pH 8.0 (Amresco E199)
• Tween 20 (Amresco 0777)
• 5M NaCl
• Fermentas ladder(s) (Ultra-low range: Fisher # FERSM1211, 50 bp: FERSM0371)
• Rare-earth magnet stand (Ambion AM10055 or NEB S1506S)

*Alternatives for Magnetic rack*
• Agencourt SPRIPlate Super Magnet Plate (Beckman Coulter A32782)
• DynaMag™-96 Side Magnet (ThermoFisher 12331D) or any
• **Add Ebay versions here.**

---

**Procedure**

1. In a 50 mL conical using sterile stock solutions, prepare TE (10 mM Tris-HCl, 1 mM EDTA = 500μL 1M Tris pH8 + 100 μL 0.5 M EDTA, fill conical to 50 mL mark with dH₂0).

2. Mix Sera-mag beads and transfer 1mL to a 1.5mL microtube.

3. Place beads on magnet stand until beads are drawn to magnet.

4. Remove supernatant with P200 or P1000 pipetter.

5. Add 1mL TE to beads, remove from magnet, mix, return to magnet.

6. Remove supernatant with P200 or P1000 pipetter.

7. Add 1mL TE to beads, remove from magnet, mix, return to magnet.

8. Remove supernatant with P200 or P1000 pipetter.

9. Add 1mL TE to beads and remove from magnet. Fully resuspend and set microtube in rack (i.e. not on magnet stand).

10. Add 9g PEG-8000 to a new 50mL, sterile conical.

11. Add 10mL 5M NaCL (or 2.92g) to conical.

12. Add 500μL 1M Tris-HCL to conical.

13. Add 100μL 0.5M EDTA to conical.

14. Fill conical to ~49mL using sterile dH20. You can do this by eye, just go slowly.

15. Mix conical for about 3-5 minutes until solution, upon sitting, is clear.

16. Add 27.5μL Tween 20 to conical and mix gently.

17. Mix 1 mL beads + TE solution and transfer to 50 mL conical.

18. Fill conical to 50 mL mark with dH₂0 (if not already there) and gently mix 50mL conical until brown.

19. Test against AMPure XP using aliquots of ladder (Fermentas GeneRuler). We recommend the 50 bp ladder in place of the ultra-low range ladder.

20. Wrap in tinfoil (or place in dark container) and store at 4°C.

:::warning
:warning: Test your beads monthly – see **Testing** below!
:::

You may also wish to prep an extra 50 mL of PEG solution that lacks Sera-mag SpeedBeads so that you can use it in a bead-inclusive library preparation protocol. You can either use the recipe from above, but leave out the beads & fill to 50 mL or you can use the recipe below, derived from Fisher (2011):

1. Add 10g PEG-8000 to a new 50mL, sterile conical.
2. Add 25mL 5 M NaCL (or 7.3g) to conical.
3. Fill conical to ~ 49 mL using sterile dH20. You can do this by eye, just go slowly.
4. Mix conical for about 3-5 minutes until PEG goes into solution (solution, upon sitting, should be clear).
5. Fill conical to 50 mL mark.

:::info
:bulb:Note: this has a slightly higher concentration of PEG (20% vs. 18%) and much higher concentration of NaCl (2.5M vs. 1M), which probably slightly changes the size-range of fragments recovered (though we tend to use them interchangeably).
:::

**Suggested Reading**

DeAngelis MM, Wang DG, Hawkins TL: [Solid-phase reversible immobilization for the isolation of PCR products](https://academic.oup.com/nar/article-abstract/23/22/4742/1065720?redirectedFrom=fulltext). ***Nucleic Acids Res.*** 1995, 23:4742–4743.

Fisher S, et al.: [A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries](https://genomebiology.biomedcentral.com/articles/10.1186/gb-2011-12-1-r1). ***Genome Biol 2011***, 12:R1.

Lundin S, Stranneheim H, Pettersson E, Klevebring D, Lundeberg J: [Increased throughput by parallelization of library preparation for massive sequencing](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0010029). ***PLoS One 2010***, 5:e10029.