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  • Size Selection of Adaptor-ligated DNA (E7530)


    Symbols 

     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.

    Protocol

    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.
    1. Vortex AMPure XP beads to resuspend.

    2. Adjust the final volume after ligation by adding nuclease free water for a 100 μl total volume.

    3. 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.

    4. Incubate for 5 minutes at room temperature.

    5. 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.

    6. Add 20 μl resuspended AMPure XP beads to the supernatant, mix well and incubate for 5 minutes at room temperature.

    7. 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).

    8. 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.

    9. Repeat Step 8 twice for a total of three washes.

    10. Air the dry beads for 10 minutes while the tube is on the magnetic stand with the lid open.

    11. 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. 
    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 insertsizes > 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.