For AMPure XP bead size selection, use the protocol below. Expect size distributions in the range of 230–270 for 100 bp reads and 310–370 for 200 bp reads.
Table 1: Recommended Conditions for Dual Bead-based Size Selection
||INSERT SIZE (bp)
|1st Bead Selection
|2nd Bead Selection
*Bead:DNA ratio is calculated based on the original volume of DNA solution.
The following protocol is for size selecting libraries with a 100 bp insert from a 100 μl volume. For libraries with a 200 bp insert, please use the bead:DNA ratio listed in Table 1.1.
AMPure XP Bead-based Dual Bead Size Selection for 100 bp Inserts
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 55 μl 0.1X TE to the adaptor ligated DNA from Section 1.2, Step 4 to bring the total volume to 100 μl.
- 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.
2nd Bead Selection to Remove Small Fragments and to Bind DNA Target: 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 (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.
- Repeat Step 7 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 0.1X TE. Mix well on a vortex mixer or by pipetting up and down, and incubate for 2 minutes at room temperature.
- Put the tube in a magnetic rack until the solution is clear. Transfer approximately 40 μl of the supernatant to a clean tube.
- Proceed to PCR Amplification of Adaptor Ligated DNA.