AMPure XP PCR Clean Up Protocol
SPRI beads are used to selectively bind DNA fragments that are greater than 100 bp. This step will remove products 100 bp and less.
AMPURE XP Bead Volume Calculations (example)
| Volumes for 18S (1.2x) | Volumes for COI (0.9x) | Volumes for 12S (0.9x) | Volumes for 16S (0.9x) | |
|---|---|---|---|---|
| Volume of amplicons | 65ul | 65ul | 40ul | 65ul |
| Volume of XP beads | 78ul | 58.5ul | 36ul | 58.5ul |
Before Starting
- First, fully resuspend the AMPure XP beads by vortexing (if no vortexer; invert the reagent bottle several times), remove 2.5 to 3.0 ml aliquot from bottle to small collection tube.
- Make fresh 80% ethanol each time
Bead Clean-up Steps
- Move the desired volume of PCR product to a new 96 well plate. Use this volume to calculate the volume of beads needed for each marker (See example in the table above)
- Add the appropriate volume of beads (1.2X or 0.9X) Ampure XP beads to combined PCR amplicons. See calcs above. Vortex to mix well (seal plate if vortexing), or pipette up and down at least 10 times. Color in wells should be homogenous
- Incubate the solution for 10 mins at room temperature to bind fragments larger than 100 bp (your amplicons) to the beads
- Place the tube on the magnetic rack to separate the beads from the supernatant and incubate until the solution becomes clear ( > 5 minutes)
- Keeping the plate/tube on the magnet, carefully remove the supernatant, leaving ~5 μL of the supernatant behind. The supernatant contains fragments less than 100 bp and you will discard.
- It is critical not to transfer beads with the supernatant because your amplicons are bound on the beads
- Keeping the plate/tube on the magnet, add 200 μl of 80% ethanol
- Keeping the plate/tube on the magnet, Incubate the plate/tube at room temperature for 1 min
- Keeping the plate/tube on the magnet, Carefully remove and discard the ethanol.
- Keeping the plate/tube on the magnet, add 200 μl of 80% ethanol
- Keeping the plate/tube on the magnet, Incubate the plate/tube at room temperature for 1 min
- Keeping the plate/tube on the magnet, Carefully remove and discard the ethanol.
- Try to remove all the residual ethanol without disturbing the beads
- Keeping the plate/tube on the magnet, Dry the beads at room temperature, until all of the ethanol has evaporated ~ 5 minutes
- Depending on the magnets used and the ambient temperature, beads may dry quicker than 5 minutes. It is important to not over-dry the beads and product can be lost. If Cracks begin to form, immediately move on the re-suspension in Qiagen Buffer EB (Elution Buffer)
- Remove the plate/tube from the magnet
- Resuspend the beads in 42.5 μL of Qiagen Buffer EB (pH 8.5). Vortex to mix well (seal plate if vortexing), or pipette up and down at least 10 times
- If a more concentrated product is desired, the volume of elution buffer can be reduced
- Incubate the plate/tube at room temperature for 5 min to elute DNA off the beads
- Place the plate/tube on a magnet to capture the beads. Incubate until the liquid is clear, ~5 mins
- Cleaned PCR product is now in the supernatent
- Transfer size-selected DNA to a new tube/96 well plate