For libraries with different size fragment inserts, refer to Table 1 for the
appropriate volume of beads to be added. The size selection protocol is
based on a starting volume of 100 μl. The protocol below is for libraries
with a 300–450 bp insert size.
Vortex AMPure XP beads to resuspend.
Adjust the final volume after ligation by adding nuclease free water for a
100 μl total volume.
Add 40 μl of resuspended AMPure XP beads to the 100 μl ligation
reaction. Mix well by pipetting up and down at least 10 times.
Incubate for 5 minutes at room temperature.
Quickly spin the tube and place the tube on an appropriate magnetic stand
to separate the beads from the supernatant. After the solution is clear
(about 5 minutes), carefully transfer the supernatant containing your DNA
to a new tube
(Caution: do not discard the supernatant). Discard the
beads that contain the unwanted large fragments.
Add 20 μl resuspended AMPure XP beads to the supernatant, mix well and
incubate for 5 minutes at room temperature.
Quickly spin the tube and place it on an appropriate magnetic stand to
separate the beads from the supernatant. After the solution is clear (about
5 minutes), carefully remove and discard the supernatant that contains
unwanted DNA. Be careful not to disturb the beads that contain the desired
(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 8 twice for a total of three washes.
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 into 25 μl of 10 mM Tris-HCl or
0.1 X TE, pH 8.0. Mix well on a vortex mixer or by pipetting up and down.
Quickly spin the tube and place it on a magnetic stand. After the solution is
clear (about 5 minutes), transfer 20 μl to a new PCR tube for amplification.
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 NEBNext
High-Fidelity 2X PCR Master Mix in the subsequent PCR step
Recommended size selection conditions for libraries with insert sizes > 300 bp.
RNA libraries made from Universal Human Reference Total RNA (500 ng) and size selected using different bead/DNA rations as indicated in Table 1. RNA was fragmented at 94°C for 5 minutes.