Protocol #03
Amplicon purification
Using AMPure beads to purify and size-select PCR amplicons
Prep:
 01 Bring AMPure beads to RT before starting the purification (30 minutes).
 02 Prepare fresh 80% EtOH. You will need approximately 0.5 mL per each sample.
 03 Vortex the AMPure beads for at least 60 seconds.
 04 Combine the PCR reaction and the AMPure beads. Per 25 µL of PCR reaction you
need to use 20 µL of AMPure beads. Mix properly by pipetting or vortexing.
 05 Incubate at RT for 5 minutes. Off the magnet.
 06 Put the samples on the magnet. Incubate for 2 minutes.
 07 Keep the samples on the magnet and remove the supernatant. Be careful not to
damage the pellet.
 08 Add 200 µL of 80% EtOH. Incubate for 30 seconds. Remove the ethanol/supernatant.
 09 Repeat the previous step: Add 200 µL of 80% EtOH, wait 30 seconds, remove the EtOH.
 10 Close the tubes/put foil on the plate. Do quick spin to collect the remaining ethanol.
 11 Put the samples back on the magnet. Wait a minute. Remove the residual ethanol with
a 10 µL pipette. Be careful not to damage the pellet.
The goal is to remove as much ethanol as possible – but the pellet cannot dry up!
 12 Remove the samples from the magnet.
 13 Add 32 µL of Elution buffer.
 14 Close the tubes/put foil on the plate. Mix the samples properly. Vortex. Quick spin.
 15 Incubate at RT for 5 minutes. Off the magnet.
 16 Put the samples on the magnet. Incubate for 2 minutes.
 17 Transfer 30 µL of the supernatant into a clean tube/plate. Be careful not to damage the
pellet. You can use the 10 µL three times to minimise the bead contamination.