AMPure Mods: Difference between revisions
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Our group uses AMPure beads to clean Illumina DNA-seq and RNA-seq libraries, as there are many advantages to using SPRI beads over other products (see Quail et al., 2009). The biggest advantage is that conditions can be modified to selectively 'lose' small DNAs in the size range of 50 - 200 bp, as is shown in this image. This can be advantageous if Illumina libraries contain adapter dimers or other contaminating oligonucleotides. While we use AMPure beads in our laboratory, this is not an endorsement of AMPure per se; similar results can be obtained using other paramagnetic SPRI beads.'' | Our group uses AMPure beads to clean Illumina DNA-seq and RNA-seq libraries, as there are many advantages to using SPRI beads over other products (see Quail et al., 2009). The biggest advantage is that conditions can be modified to selectively 'lose' small DNAs in the size range of 50 - 200 bp, as is shown in this image[[Image:AMpure_purification_08312010.jpg|thumb|right|Gel image showing removal of adapter]]. This can be advantageous if Illumina libraries contain adapter dimers or other contaminating oligonucleotides. While we use AMPure beads in our laboratory, this is not an endorsement of AMPure per se; similar results can be obtained using other paramagnetic SPRI beads.'' | ||
Revision as of 18:28, 22 November 2010
Our group uses AMPure beads to clean Illumina DNA-seq and RNA-seq libraries, as there are many advantages to using SPRI beads over other products (see Quail et al., 2009). The biggest advantage is that conditions can be modified to selectively 'lose' small DNAs in the size range of 50 - 200 bp, as is shown in this image
. This can be advantageous if Illumina libraries contain adapter dimers or other contaminating oligonucleotides. While we use AMPure beads in our laboratory, this is not an endorsement of AMPure per se; similar results can be obtained using other paramagnetic SPRI beads.
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
• DNA seq AMPure Sample+Water 45 µl 65 µl 90 µl 130 µl
• RNA seq AMPure Sample+Water 45 µl 55 µl 90 µl 110 µl
Abbreviated protocol for AMPure bead purification
• Gently shake AMPure bottle to resuspend magnetic particles that have settled.
• Add the desired volume of Agencourt AMPure beads
• Incubate samples+water + AMPure at room temperature for 5 minutes.
• Magnetize and then incubate for 5 minutes.
• Aspirate the cleared supernatant and discard.
• Dispense 200 µl of 80% EtOH 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 to air dry for 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 5 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 over try beads in the spin-vac or PCR machine; air-temp drying is preferred.
