General Guidelines for AMPure-bead based isolation
• Illumina DNA-Seq libraries: Use 0.7 volumes AMPure to 1.0 volume sample. Eliminates 100bp and 200bp bands, retains > 300bp bands
• Illumina RNA-Seq libraries: Use 0.9 volumes AMPure to 1.0 volume sample. Eliminates 100bp band, retains > 200bp bands
• Routine DNA and PCR cleaning: Use 1.5 volumes AMPure to 1.0 volume sample. Eliminates primers and small oligos
Example volumes for DNA-seq and RNA-seq libraries
• for DNA seq
AMPure : Sample+Water = 45 µl AMPure : 65 µl Sample+Water
• for RNA seq
AMPure : Sample+Water = 45 µl AMPure : 55 µl Sample+Water
Abbreviated protocol for AMPure bead purification
• Prior to use, remove the beads from storage and let them stand for 30 minutes to warm to room temperature.
• Just before use, vortex or shake the AMPure bottle to resuspend magnetic particles that have settled. The suspension should appear completely homogenous.
• Add the desired volume of Agencourt AMPure beads. Mix the solution ten times by pipetting up and down.
• Incubate samples + water + AMPure at room temperature for 15 minutes for maximum recovery (5 minutes for decent yield).
• Magnetize and then incubate for at least 5 minutes (more time may be required for viscous solutions).
• Aspirate the cleared supernatant SLOWLY and discard. You can leave up to 5 ul of supernatant remaining in the tube at this step.
• Dispense 200 µl of 80% EtOH (freshly prepared every time) into each tube and incubate for 30 seconds. Aspirate the EtOH wash and discard. Repeat for a total of two washes.
• Remove all traces of EtOH with a fine pipette tip, and place on bench until completely air dried. We recommend at least 10 minutes.
• Add the desired volume of elution buffer (10 mM Tris-HCl, pH 8.0 or water) and vortex for 15-30 seconds.
• Incubate the samples at room temperature for at least 2 minutes.
• Magnetize the sample one last time for 5 minutes.
• Aspirate the supernatant (the ELUTED DNA) and transfer to a new tube.
TIPS!!! Don’t let the beads go dry between wash steps. Don’t dry beads in the spin-vac or PCR machine; air-temp drying is preferred.
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