Size Selection Using AMPure XP Beads - NEBNext Fast DNA Library Prep Set for Ion Torrent (E6270)
Table 1: Recommended Conditions for Dual Bead-based Size Selection
|BEAD:DNA RATIO*||INSERT SIZE (bp)|
|100 bp||200 bp|
|1st Bead Selection||0.9X||0.7X|
|2nd Bead Selection||0.15X||0.15X|
AMPure XP Bead-based Dual Bead Size Selection for 100 bp Inserts
The following size selection protocol is for libraries with 100 bp insert from a 100 μl volume. For libraries with a 200 bp insert please use the bead:DNA ratio listed in the chart above
1st Bead Selection to Remove Large Fragments:
This step is used to bind the large, unwanted fragments to the beads. The supernatant will contain the desired fragments.
- Add 90 µl (0.9X) resuspended AMPure XP beads to 100 µl DNA solution. Mix well on a vortex mixer or by pipetting up and down at least 10 times.
- Incubate for 5 minutes at room temperature.
- Place the tube on a magnetic rack to separate the beads from the supernatant. After the solution is clear (about 5 minutes), carefully transfer the supernatant to a new tube (Caution: do not discard the supernatant) Discard beads that contain the large fragments.
This step will bind the desired fragment sizes (contained in the supernatant from Step 3) to the beads. Unwanted smaller fragment sizes will not bind to the beads.
- Add 15 µl (0.15X) resuspended AMPure XP beads to the supernatant, mix well and incubate for 5 minutes at room temperature.
- Put the tube on a magnetic rack to separate beads from supernatant. After the solution is clear (approximately 3 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.
- Repeat Step 6 once.
- Keeping the tube on the magnetic rack, with the cap open, air dry the beads for 5 minutes.
Caution: Do not overdry the beads. This may result in lower recovery of DNA target.
- Remove the tube from the magnet. Elute DNA target from beads into 42 μ l sterile 0.1X TE. Mix well on a vortex mixer or by pipetting up and down, incubate for 2 minutes at room temperature.
- Put the tube in a magnetic rack until the solution is clear, approximately 3 minutes. Transfer approximately 40 μl of the supernatant to a clean tube.
- Proceed to PCR Amplification.