How do I remove long fragments from a library?

Ampure XP/SPRI bead “upper cut” protocol to remove double-stranded DNA fragments over 670 bases:
Bead-based size selections are the preferred method as they can be applied in a high-throughput fashion to your samples.  Please note:
  • Bead-based size selection cannot carry out precise “cuts”;  Thus, you will also lose some of the library molecules in the size ranges that you intend to keep. This selection protocol will also reduce adapter dimers and other molecules shorter than 160 bp.
  • It is recommended to verify this protocol first with your batch of beads.
  • Multiple other manufacturers offer copies of the Ampure XP product (e.g. SPRI beads, Kapapure, …). These can work just as efficiently. Please test them beforehand.
  • The cutoff fragment length can be modified by changing the ratios of SPRI-beads to sample volume.
      1. If not mentioned explicitly follow the standard Ampure XP handling extractions from the manufacturer (e.g. equilibrate the beads to room temperature before use; vortex beads before use, details of the bead washes and elution,…)
      2. If the sample volume is smaller than 50 microliters, add molecular biology grade water up to 50 microliters.
      3.  Add 0.55x the sample volume in Ampure XP beads to your sample, mix, incubate for 5 minutes at RT.
      4. Collect the beads on a magnet.
      5. Transfer the supernatant to a new tube.
      6. Add another 1x original volume Ampure beads to the supernatant; mix; incubate for 5 minutes
      7. Collect the beads on a magnet and remove the supernatant
      8. Carry out the regular 80% ethanol washes of the beads and elute the samples from the beads according to Agencourt Ampure XP protocol.
      9. Verify the success of the size selection by running an aliquot on a Bioanalyzer or equivalent instrument.

Category: 04 Library Preparation and QC

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