Purification of pooled PCR amplicon libraries using SPRI beads
Vortex AMPure XP or RXN pureplus SPRI beads to resuspend .
Make your pooled library up to 100 μl if necessary .
Add 60 μl resuspended SPRI beads to 100 μl of pooled PCR product .
Mix well by pipetting up and down at least 10 times .
Vortex AMPure XP beads to resuspend .
Incubate for 5 minutes at room temperature .
Quickly spin the tube and place it on an appropriate magnetic stand to separate beads from supernatant .
After the solution is clear ( about 5 minutes ) , carefully remove and discard the supernatant .
Be careful not to disturb the beads that contain DNA targets ( Caution : do not discard beads ) .
Add 200 μl of 80 % freshly prepared ethanol to the tube while in the magnetic stand .
Incubate at room temperature for 30 seconds , and then carefully remove and discard the supernatant .
Add 200 μl of 80 % freshly prepared ethanol to the tube while in the magnetic stand .
Incubate at room temperature for 30 seconds , and then carefully remove and discard the supernatant .
Add 200 μl of 80 % freshly prepared ethanol to the tube while in the magnetic stand .
Incubate at room temperature for 30 seconds , and then carefully remove and discard the supernatant .
Air the dry beads for 10 minutes while the tube is on the magnetic stand with the lid open .
Elute the DNA target from the beads by adding 30 μl of 10 mM Tris - HCl , pH 8.0 or 0.1X TE . Note : Be sure not to transfer any beads .
Trace amounts of bead carry over may affect the optimal performance of the polymerase used in the subsequent PCR step .
Mix well by pipetting up and down , or on a vortex mixer .
Quickly spin the tube and place it on the magnetic stand .
After the solution is clear ( about 5 minutes ) , transfer 27ul a new PCR tube for amplification .
