Cleanup of PCR Amplification (E7370)
Protocol
- Vortex AMPure XP beads to
resuspend.
- Add 50 μl of resuspended AMPure XP beads
to the PCR reactions (~ 50 μ l). Mix well by pipetting up and down at least 10
times.
- 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% ethanol to the PCR plate
while in the magnetic stand. Incubate at room temperature for 30 seconds, and
then carefully remove and discard the supernatant.
- Repeat Step 5 once.
- Air dry the beads for 10 minutes while the
PCR plate is on the magnetic stand with the lid open.
- Elute DNA target from beads into 33 μl 10
mM Tris-HCl, pH 8.0 or 0.1X TE. Mix well by pipetting up and down at least 10
times. 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 transfer 28 μl supernatant to a new PCR tube. Store libraries at
–20°C.
- Dilute the library 5 fold with water, and check the size distribution on an Agilent high sensitivity chip.
