Purify the Double-stranded cDNA Using 1.8X Agencourt AMPure XP Beads
This is a point where you can safely stop the protocol and store the samples prior to proceeding to the next step in the protocol.
- Vortex AMPure XP beads to
- Add 144 μl (1.8X) of resuspended AMPure XP
beads to the second strand synthesis reaction (~80 μl). Mix well on a vortex
mixer or by pipetting up and down at least 10 times.
- Incubate for 5 minutes at room
- Quickly spin the tube in a microcentrifuge
to collect any sample on the sides of the tube. Place the tube on an appropriate
magnetic rack 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.
- Add 200 μl of freshly prepared 80% ethanol
to the tube while in the magnetic rack. Incubate at room temperature for 30
seconds, and then carefully remove and discard the supernatant.
- Repeat Step 5 once for a total of 2 washing steps.
- Air dry the beads for 10 minutes while the
tube is on the magnetic rack with lid open.
- Elute the DNA target from the beads into
60 μl nuclease-free water. Mix well on a vortex mixer or by pipetting up and
down. Quickly spin the tube and then place it in the magnetic rack until the
solution is clear.
- Remove 55.5 μl of the supernatant and transfer to a clean nuclease-free PCR tube.