Ampure XP bead “upper cut” protocol to remove fragments longer than 670 bases:
-
-
- If not mentioned explicitly follow the standard Ampure XP handling instructions from the manufacturer (e.g. equilibrate the beads at to room temperature before use; vortex beads before use, details of the bead washes and elution,…)
- If the sample volume is smaller than 50 ul, add EB buffer up to 50 ul to each sample.
- Add 0.55x the sample volume in Ampure beads (e.g. 27.5 ul beads to a 50 ul sample) to your sample, mix, incubate for 5 minutes at RT.
- Collect the beads on a magnet.
- Transfer the supernatant to a new tube.
- Add another 1x original volume Ampure beads to the supernatant; mix; incubate for 5 minutes
- Collect the beads on a magnet and remove the supernatant
- Carry out the two regular 80% ethanol washes of the beads and elute the samples from the beads according to Agencourt Ampure XP protocol.
- Verify the success of the size selection by running an aliquot on a Bioanalyzer or equivalent instrument.
-
Ampure XP bead “upper & lower cut” protocol to remove fragments longer than 670 bases and shorter than 400 bases:
This protocol is identical to the one above but adds a smaller volume of the beads at step 6 for the final enrichment onto the beads. The reduced bead buffer concentration at this step leads to a removal of longer fragments compared to the protocol above.
Please note: It is recommended to verify this protocol first with your batch of Ampure XP beads or similar beads from other manufacturers. Bead-based size selection cannot carry out precise “cuts”; thus, you will also lose some of the library in the size ranges that you intend to keep.
- If not mentioned explicitly follow the standard Ampure XP handling instructions from the manufacturer (e.g. equilibrate the beads at to room temperature before use; vortex beads before use, details of the bead washes and elution,…)
- If the sample volume is smaller than 50 ul, add EB buffer up to 50 ul to each sample.
- Add 0.55x the sample volume in Ampure beads (e.g. 27.5 ul beads to a 50 ul sample) to your sample, mix, incubate for 5 minutes at RT.
- Collect the beads on a magnet.
- Transfer the supernatant to a new tube.
- Add another 0.25x of the original volume Ampure beads (e.g. 12.5 ul beads for a sample of a 50 ul starting volume sample) to the supernatant; mix; incubate for 5 minutes
- Collect the beads on a magnet and remove the supernatant
- Carry out the two regular 80% ethanol washes of the beads and elute the samples from the beads according to Agencourt Ampure XP protocol.
- Verify the success of the size selection by running an aliquot on a Bioanalyzer or equivalent instrument.
Beckmann/Agencourt also sells beads that are dedicated to size selections named SPRIselect — however, very likely these are actually identical to the AMpure XP beads. The SPRIselect manual provides a lot of additional information and protocols that can be applied to AMpure XP and other beads. Please see here: Beckman SPRIselect Ampure beads
BTW, our favorite magnetic separator for 96-well plates is this one from EdgeBio.
← FAQ