In this protocol

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Product Description

KAPA Pure Beads is a suspension of paramagnetic beads in a buffer optimized for the purification or size selection of single- and double-stranded DNA in next-generation sequencing and other molecular biology workflows. The product is designed for fast and reliable purification or size selection of 1 ng to 5 µg DNA in a single reaction. KAPA Pure Beads is compatible with manual processing or automated liquid handling and enables efficient recovery of input DNA in both formats.

For purification and size selection of DNA, the KAPA Pure Beads buffer includes PEG/NaCl—a crowding agent designed to drive DNA molecules to the beads for binding. 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.

Product Applications

KAPA Pure Beads is ideally suited for:
● fast and efficient reaction cleanups to remove adapters, adapter-dimers, primers, primer-dimers, nucleotides, salts, and enzymes in NGS library preparation, PCR, and qPCR workflows,
● size selection of fragmented input DNA, adapter- ligated library molecules, or amplified libraries in NGS library preparation workflows, and
● general manipulation of DNA samples, e.g., sample concentration and buffer exchange (e.g., from salts, buffer components, enzymes, etc.).

Protocols

  1. Genomic DNA Purification (Cleanup)
    Prior to library construction in NGS workflows, it may be beneficial to perform an upfront genomic DNA cleanup. For cleanup, buffer exchange, and/ or concentration of high-quality genomic DNA prior to library construction, a KAPA Pure Beads- to-sample volumetric ratio of 3X is 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. The heated elution is not required for the cleanup, purification, or buffer exchange of other DNA types, e.g., fragmented DNA, NGS libraries or amplicons.

    Ensure that the plate/tube(s) selected for the cleanup can accommodate the DNA sample plus the appropriate volume of KAPA Pure Beads, and that it is compatible with your magnet and heating device.
    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 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 2 °C to 8 °C for 1 – 2 weeks, or at -15 °C to -25 °C.

  2. Cleanup of Fragmented DNA in NGS Workflows
    In NGS library construction workflows, the appropriate bead-to-sample ratio depends on the point in the workflow at which the cleanup is performed (e.g., after fragmentation, adapter ligation, or library amplification), and the desired fragment sizes to be retained/excluded. KAPA Pure Beads may be employed for the effective cleanup of fragmented DNA at various stages of NGS library preparation workflows.

    The size range of DNA fragments recovered during a single-sided bead-based cleanup is dependent on the volume (ratio) of KAPA Pure Beads added to the DNA sample. For fragmented DNA, NGS libraries, and amplicons, recommendations for KAPA Pure Beads-to-sample volumetric ratios based upon desired fragment lengths to be retained are provided in Table 2 (shown in Figure 1), and should be used as a guideline.

    The detailed protocol outlined below is an example of a 0.8X cleanup of a 100 µL fragmented DNA sample.
    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 2°C to 8°C for 1 – 2 weeks, or at -15°C to -25°C.

    Table 2: Guidelines for the purification (cleanup) of fragmented DNA, NGS libraries, and amplicons with KAPA Pure Beads



    Figure 1. Effect of KAPA Pure Beads-to-sample ratio on the recovery of dsDNA fragments. A defined mixture of dsDNA fragments (ranging from 100 bp to 10 kb) was processed using various KAPA Pure Beads-to-sample ratios. The impact on DNA fragment size retention was assessed with an Agilent® Bioanalyzer 2100 High Sensitivity DNA Kit. Retained DNA fragment lengths are inversely proportional to the bead-to-sample ratio; a greater volume or ratio of beads is required to retain lower molecular weight fragments.

  3. Size Selection in NGS Workflows
    Size selection requirements vary widely for different sequencing applications. KAPA Pure Beads may be integrated into most DNA library construction workflows, and size selection can be carried out at various points in the overall workflow (e.g., after fragmentation, post-ligation cleanup, or library amplification).

    Guidelines for size selection of Illumina libraries with KAPA Pure Beads are given in Table 3, and representative traces of size-selected input DNA and libraries are given in Figure 2. These parameters are provided as guidelines only and may require additional optimization.

    The following detailed protocol is an example of size selection of adapter-ligated library in a 50 µL volume. As per Table 3, a 0.6X – 0.8X size selection is used to target a final library size distribution of 250 – 450 bp. The first 0.6X cut is designed to exclude molecules > 450 bp from the library-containing supernatant retained for the second cut. The additional 0.2 volumes of KAPA Pure Beads results in the binding of all molecules > 250 bp (but < 450 bp) to the beads. DNA fragments < 250 bp are discarded with the supernatant during the bead washes.

    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 2°C to 8°C for 1 – 2 weeks, or at -15°C to -25°C.

    Table 3: Guidelines for size selection with KAPA Pure Beads

    1Adapter ligation increases the length of DNA fragments. To achieve the same final fragment size, a lower ratio of KAPA Pure Beads is therefore required for the first cut when performing size selection after ligation or library amplification, as opposed to after fragmentation. Please refer to Figure 2 for more details.
    2These size selection parameters were optimized for libraries prepared with single-indexed, Illumina TruSeq-style adapters. Parameters have to be optimized for libraries prepared with dual-indexed adapters, shorter or custom adapter designs.
    3When performing size selection after ligation, it is important to perform at least one post-ligation cleanup, as KAPA Ligation Buffers contain high concentrations of PEG 6000, which will interfere with size selection (double-sided cleanups) if not removed.


    Figure 2. Examples of input DNA and NGS libraries subjected to bead-based size selection using KAPA Pure Beads at different stages of the library construction process.
    A. Size distributions of fragmented input DNA (no size selection), vs. final, amplified libraries prepared from the same DNA, but size selected after fragmentation or after ligation, respectively. Since the mode fragment size of input DNA increases after adapter ligation, the KAPA Pure Beads-to-sample volumetric ratio used for the first cut during post- ligation size selection was lower than for post-fragmentation size selection to target the same final mode library size distribution (300 – 750 bp).
    B. Size distributions for final, amplified libraries that were not size selected vs. libraries prepared from the same input DNA, but size selected with different parameters after the post-ligation cleanup to achieve different final size distributions (as outlined in Table 2).
    All libraries were prepared with the KAPA Hyper Prep Kit, from 100 ng high-quality human genomic DNA, fragmented with a Covaris E220 Focused Ultrasonicator using conditions optimized to yield a mode fragment length in the range of 350 – 450 bp. Electropherograms were generated with a Bioanalyzer 2100 High Sensitivity DNA Kit. DNA concentrations were normalized prior to analysis and are not reflective of actual concentrations at different stages of the library preparation workflow.

Troubleshooting

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