• sunbet(中国)

    Bead clean-up (single tube)

    timothy.creed

    Published: 2023-02-07 DOI: 10.17504/protocols.io.n92ldz5qxv5b/v1
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    Abstract

    Protocol for purification of DNA using SPRI beads

    Steps

    Prepare reagents

    1.

    Prepare the following reagents/materials:

    or Homemade SPRI beads

    Fresh 70% Ethanol

    Nuclease-free water or elution buffer

    DNA sample/PCR product

    Clean 1.5 mL tubes

    Magnetic tube rack

    Note
    - Make sure beads are thoroughly mixed/vortexed to ensure they are well resuspended, the solution should be a homogenous brown color.- Beads and sample must be brought to Room temperature before mixing.

    Note
    - Ethanol should be freshly prepared. Due to miscibility of EtOH and water, prepare by adding volumetric parts, not by bringing to volume.

    Add beads to sample

    2.

    In a 1.5 mL tube, add resuspended beads to sample/PCR product in ratio specified in main protocol.

    Note
    The ratio is dependent on the length of DNA you want to recover.

    2.1.

    Flick tube gently to mix beads and sample.

    Note
    Pipette mixing may fragment the sample. Do not vortex to mix.

    2.2.

    Allow mixture to incubate for 0h 5m 0s at Room temperature

    Note
    Place tube on slow rotation mixer or flick gently periodically throughout the incubation to prevent beads from settling to the bottom of the tube.

    Separation

    3.

    Place tube on a magnetic rack.

    Equipment

    ValueLabel
    Magnetic StandNAME
    Magnetic StandTYPE
    Thermo ScientificBRAND
    MR02SKU
    Any magnetic rack that fits your tubes will suffice.SPECIFICATIONS
    3.1.

    Wait ~1-3 minutes for the beads and buffer to separate. Beads will stick to magnetic side of the tube and the solution should be clear.

    3.2.

    Using a pipettor, remove and discard the clear supernatant solution. DNA will remain in the tube bound to the beads.

    Ethanol Washing

    4.

    Add 500 μL 70% EtOH to tube

    • Do not disturb the beads! Pipette into the opposite side of the tube.
      Note
      Volume of 70% EtOH can be adjusted. Just so long as the amount added is enough to cover beads.
    4.1.

    Incubate at Room temperature for 0h 0m 30s

    4.2.

    Using a pipettor, remove and discard the EtOH. Be careful not to disturb or aspirate the beads.

    5.

    Repeat the Step 4 EtOH wash. Try to remove as much EtOH as possible without disturbing the beads.

    Note
    A quick spin-down may help to remove residual EtOH.

    Air dry beads

    6.

    Leaving tube cap open, allow beads to dry for a maximum of 90 seconds.

    Note
    -Do not over-dry beads. Over-dried beads do not resuspend well and can lead to a loss of DNA. -If the surface of beads appear cracked, they are over-drying. Resuspend immediately.

    Resuspend

    7.

    Remove tube from magnetic rack and resuspend DNA/beads by adding a minimum of 15 μL nuclease-free water or desired buffer.

    Note
    The exact resuspension volume should be specified in your main protocol. Use a smaller volume to increase concentration of DNA

    7.1.

    Flick tube gently to resuspend beads in elution.

    Incubate

    8.

    Incubate at Room temperature for 0h 2m 0s- 0h 5m 0s

    Recovery

    9.

    Place tube back on magnetic rack. Allow beads and elution to separate for 0h 1m 0s .

    9.1.

    Slowly aspirate out clear supernatant, now containing DNA, and transfer to final clean container.

    Note
    Do not carry over any beads. They are a significant inhibitor of various downstream applications. Carefully check your pipette tip for bead carryover. If there are beads, replace the sample into the tube and wait another 2 min. You may need to decrease the volume removed from the tube by 1-2 μL.

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