SPRI Bead Cleanup of DNA

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Overview

This is a protocol to cleanup, purify, and/or concentrate DNA samples using solid-phase reversible immobilization (SPRI) beads. AMPure XP beads are used in the author's lab, but any old SPRI beads should do.

Note: This protocol is far from optimized, and has been designed to capture a 4.5 kb DNA fragment. The ratio of beads to volume of DNA-containing solution must be adjusted depending on the length of the DNA fragment of interest.

Materials

  • Room Temperature SPRI Beads
  • Fresh 80% Ethanol
  • Nuclease-Free Water

Equipment

  • Something with which to aspirate would be helpful, i.e. vacuum line hooked up to a sidearm flask. A pipette can also be used, with care

Procedure

  1. Add 2:1 volume of beads to each PCR tube containing DNA solution.
  2. Vortex to mix, then quickly spin down the PCR tubes.
  3. Incubate at room temperature for 10 minutes.
  4. Place tubes on magnetic rack for 5 minutes.
  5. Aspirate the liquid in each tube.
  6. Being careful not to disturb the beads, add 200 μL of fresh 80% ethanol. Wait 30 seconds, then aspirate.
    1. Repeat the above wash, this time getting as much ethanol as possible. A drying period may be necessary. Not only will residual ethanol interfere with downstream applications, but it will reduce yield by effecting interactions between the DNA and beads.
  7. Resuspend beads in nuclease-free water.
    1. Here, I use 20 μL per tube, but this can be adjusted depending on anticipated concentration and downstream application.
  8. Incubate away from the magnet for 10 minutes.
  9. Incubate on the magnet for 5 minutes.
  10. Carefully remove the supernatant, and combine all aliquots into one 1.5 mL tube. Qubit to measure DNA concentration.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  • Eluting in TE might be okay, but the salts and EDTA may mess with the beads. Someone smarter than me knows for sure.
  • This can also be applied to RNA cleanup, but care must be taken to avoid RNase contamination.


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