Magnetic Bead Wash
The protocol for doing a magnetic bead wash.
Overview
Magnetic beads are used for purification and size selection of single or double-stranded DNA. size selection may be performed at the following points during the overall library construction process:
before or after DNA fragmentation;
after individual enzymatic reactions;
after the first post-ligation cleanup; or
after library amplification.
We use Kapa Pure Beads for magnetic bead washes. We find that they work as well as the Ampure Beads and are MUCH cheaper.
Before using, be sure to read the PDF from Kapa.
Table of Contents
Protocols
Troubleshooting
Protocols
See the PDF for the complete protocol. Tips:
Cleanup genomic DNA
1.1 Ensure that KAPA Pure Beads has been equilibrated to room temperature and that the beads are fully resuspended before proceeding.
1.2 Add 300 μL of KAPA Pure Beads (3:1 ratio for gDNA) to the 100 μL genomic DNA sample.
1.3 Mix thoroughly by vortexing and/or pipetting up and down multiple times.
1.4 Incubate the plate/tube(s) at room temperature for 10 min to bind the DNA to the beads.
1.5 Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
1.6 Carefully remove and discard the supernatant.
1.7 Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
1.8 Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
1.9 Carefully remove and discard the ethanol.
1.10 Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
1.11 Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
1.12 Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
1.13 Dry the beads at room temperature for 3 – 5 min, or until all of the ethanol has evaporated. Caution: over-drying the beads may result in reduced yield.
1.14 Remove the plate/tube(s) from the magnet.
1.15 Resuspend the beads in an appropriate volume of pre-heated elution buffer at 37°C and/or perform the elution incubation (step 1.16) in a thermocycler or heating block set to 37°C. The appropriate elution buffer may be either 10 mM Tris-HCl, (pH 8.0 – 8.5) or PCR-grade water, depending on the downstream application.
1.16 Incubate the plate/tube(s) for 10 min to elute the DNA off the beads.
1.17 Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
1.18 Transfer the clear supernatant to a new plate/ tube(s). Proceed with your downstream application, or store DNA at 4ºC for 1 – 2 weeks, or at -20ºC.
Notes
3X KAPA Pure Beads to-sample volumetric ratio recommended
The detailed protocol below is an example of a 3X cleanup of genomic DNA in 100 µL. Please pay special attention to steps 1.15 and 1.16 (elution of DNA off beads). To ensure optimal recovery, these steps may be performed at an elevated temperature: 37°C for 10 min. Elution buffer may be pre-heated for this step and/or the elution performed in a thermocycler or heating block.
As indicated above, a heated elution is recommended to ensure optimal recovery of high-quality genomic DNA. This is not a requirement for the cleanup of other DNA types (e.g. fragmented DNA).
For maximum recovery of the original DNA sample during cleanup, buffer exchange, and/or concentration of genomic DNA, fragmented DNA for NGS library construction, PCR or qPCR products—regardless of quality—a KAPA Pure Beads-to-sample volumetric ratio of 3X is recommended. Expected recoveries are 50 – 90%, depending upon sample quality and quantity. If a lower ratio is used, it may result in lower yields and exclusion of smaller fragments.
Cleanup of fragmented DNA
2.1 Ensure that KAPA Pure Beads has been equilibrated to room temperature and that the beads are fully resuspended before proceeding.
2.2 Add 80 μL of KAPA Pure Beads to the 100 μL fragmented DNA sample.
2.3 Mix thoroughly by vortexing and/or pipetting up and down multiple times.
2.4 Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind the DNA to the beads.
2.5 Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
2.6 Carefully remove and discard the supernatant.
2.7 Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
2.8 Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
2.9 Carefully remove and discard the ethanol.
2.10 Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
2.11 Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
2.12 Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
2.13 Dry the beads at room temperature for 3 – 5 min, or until all of the ethanol has evaporated. Caution: over-drying the beads may result in reduced yield.
2.14 Remove the plate/tube(s) from the magnet.
2.15 Resuspend the beads in an appropriate volume of elution buffer (10 mM Tris-HCl, pH 8.0 – 8.5) or PCR-grade water, depending on the downstream application.
2.16 Incubate the plate/tube(s) at room temperature for 2 min to elute the DNA off the beads. The elution time may be extended up to 10 min if necessary to improve DNA recovery.
2.17 Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
2.18 Transfer the clear supernatant to a new plate/ tube(s). Proceed with your downstream application, or store DNA at 4ºC for 1 – 2 weeks, or at -20ºC.
Notes
- 0.8X cleanup of a 100 µL fragmented DNA sample recommended (will retain fragments greater than 300 bp)
| Fragments to be retained | Recommended Beads-to-sample ratio |
|---|---|
| ≥ kb | 0.5X |
| ≥ 450 bp | 0.6X |
| ≥ 350 bp | 0.7X |
| ≥ 300 bp | 0.8X |
| ≥ 250 bp | 0.9X |
| ≥ 150 bp | 1.5X |
| ≥ 100 bp | 2.2X -3X |
Size Selection
3.1 Ensure that KAPA Pure Beads has been equilibrated to room temperature and that the beads are fully resuspended before proceeding.
3.2 Perform the first size cut (0.6X, to exclude large library fragments) by adding 30 μL of KAPA Pure Beads to 50 μL of adapter-ligated library (0.6 x 50 μL = 30 μL).
3.3 Mix thoroughly by vortexing and/or pipetting up and down multiple times.
3.4 Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind large library molecules (>450 bp) to the beads.
3.5 Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
3.6 Carefully transfer the supernatant containing the smaller library molecules (<450 bp) to a new plate/ tube(s). It is critical that no beads are transferred with the supernatant.
3.7 Discard the plate/tube(s) containing the beads to which library fragments larger than ~450 bp are bound.
3.8 Perform the second size cut (0.8X) by adding 10 μL of KAPA Pure Beads to the supernatant. This volume is calculated relative to the original sample volume of 50 μL, e.g., (0.8 – 0.6) x 50 μL = 10 μL.
3.9 Mix thoroughly by vortexing and/or pipetting up and down multiple times.
3.10 Incubate the plate/tube(s) at room temperature for 5 – 15 min to bind library molecules >250 bp to the beads.
3.11 Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear. Carefully remove and discard the supernatant which contains library molecules smaller than
250 bp.
3.12 Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
3.13 Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
3.14 Carefully remove and discard the ethanol. 3.15 Keeping the plate/tube(s) on the magnet, add 200 μL of 80% ethanol.
3.16 Incubate the plate/tube(s) on the magnet at room temperature for ≥30 sec.
3.17 Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
3.18 Dry the beads at room temperature for 3 – 5 min, or until all of the ethanol has evaporated. ⚠️ Caution: over-drying the beads may result in reduced yield.
3.19 Remove the plate/tube(s) from the magnet.
3.20 Thoroughly resuspend the beads in the required volume of elution buffer (10 mM Tris-HCl, pH 8.0 – 8.5), or PCR-grade water, depending on the downstream application.
3.21 Incubate the plate/tube(s) at room temperature for 2 min to elute DNA off the beads. The elution time may be extended up to 10 min if necessary to improve DNA recovery.
3.22 Place the plate/tube(s) on a magnet to capture the beads. Incubate until the liquid is clear.
3.23 Transfer the clear supernatant with size-selected DNA to a new plate/tube(s). Proceed with your downstream application, or store DNA at 4ºC for 1 – 2 weeks, or at -20ºC.
Notes
Bead-based size selection or “double-sided size selection” consists of a “first” and “second cut”, represented as ratios of beads-to-sample volumes.
Factors that Impact Overall Performance
Store at 2°C to 8°C in a constant-temperature refrigerator; do not store at -20°C. Always ensure that KAPA Pure Beads is fully equilibrated to room temperature before use.
For optimal performance, always ensure that the KAPA Pure Beads is equilibrated to room temperature, that the beads are fully resuspended before use, and that the DNA and bead solutions are thoroughly mixed prior to the binding incubation.
The volumetric ratio of KAPA Pure Beads to sample is the critical factor in determining the size distribution of DNA fragments retained by the beads. The volume (ratio) may be modified/optimized based upon the specific application and/or point in the library construction workflow where a cleanup or size selection is employed.
The volume in which cleanups and size selection are performed may impact performance. DNA samples may be diluted in 10 mM Tris-HCl (pH 8.0 – 8.5) or PCR-grade water to increase the working volume prior to the addition of KAPA Pure Beads. This is recommended for starting volumes <50 µL, DNA preparations that are viscous, and/ or contain a high concentration of salts or PEG.
It is important to prepare fresh 80% ethanol prior to use, since ethanol absorbs atmospheric water.
The volume of 80% ethanol used for bead washes may be adjusted to accommodate different plate/tube volumes and/or limited pipetting capacity, but it is important that the beads are entirely submerged during the wash steps. Where possible, use a wash volume that is equal to the volume of sample plus KAPA Pure Beads.
It is important to remove all ethanol before proceeding with subsequent reactions. However, over-drying of beads may make them difficult to resuspend, and may result in a dramatic loss of DNA. Drying of beads for 3 – 5 min at room temperature should be sufficient. Drying of beads at 37ºC is not recommended.
Elution time and temperature may impact DNA recovery, particularly for high-molecular weight genomic DNA.
Purified DNA in elution buffer should be stable at 4°C for 1 – 2 weeks, or for at least 1 month at -20°C. The long-term stability of library DNA at -20°C depends on a number of factors, including library concentration. Always use low DNA-binding tubes for long-term storage and avoid excessive freezing and thawing.
To increase the recovery of DNA at a specific stage in an NGS library construction protocol, the volume of KAPA Pure Beads used in the cleanup may be increased, but this will most likely result in the retention of smaller DNA fragments.
Upper size limit | Modification
Increase upper size limit: Decrease the ratio of the first cut
Decrease upper size limit: Increase the ratio of the first cut
Lower size limit | Modification
Increase lower size limit: Decrease the ratio of the second cut
Decrease lower size limit: Increase the ratio of the second cut
The second size cut should be performed with at least 0.2 volumes of KAPA Pure Beads. Please note the volume of the KAPA Pure Beads needed for the second cut is calculated relative to the volume of the sample at the start of the size selection procedure, not the volume of the DNA-containing supernatant transferred after the first cut. DNA recovery is dramatically reduced if the difference between the first and second cuts is less than ~0.2 volumes. To increase the amount of DNA recovered, more than 0.2 volumes of KAPA Pure Beads may be used for the second cut, but note that this may result in the recovery of smaller library fragments and a broader size distribution.