Size Selection of Adaptor-ligated DNA (E7530)
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.
This caution sign signifies a step in the protocol that has two paths leading to the same end point but is dependent on a user variable, like the type of RNA input.
ProtocolFor 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
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 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 28 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 23 μl to a new PCR tube for amplification.