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  • Purify the Ligation Reaction Using AMPure XP Beads (E7420)

    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

     Note: If you are selecting for larger size fragments (> 200 nt) follow the size selection recommendations in Appendix A on the manual.

    1. To the ligation reaction (83.5 μl), add 16.5 μl nuclease-free water to bring the reaction volume to 100 μl.
    Note: X refers to the original sample volume of 100 µl from the above step.

    1. Add 100 μl (1.0X) resuspended AMPure XP beads and mix well on a vortex mixer or by pipetting up and down at least 10 times.
    2. Incubate for 5 minutes at room temperature.
    3. Quickly spin the tube in a microcentrifuge and place the tube on an appropriate magnetic rack to separate beads from the supernatant. After the solution is clear (about 5 minutes), discard the supernatant that contain unwanted fragments (Caution: do not discard the beads).
    4. Add 200 μl of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
    5. Repeat Step 5 once for a total of 2 washing steps.
    6. Briefly spin the tube, and put the tube back in the magnetic rack.
    7. Completely remove the residual ethanol, and air dry beads for 10 minutes while the tube is on the magnetic rack with the lid open.
    8. Elute DNA target from the beads with 50 μl nuclease-free water. Mix well on a vortex mixer or by pipetting up and down, and put the tube in the
      magnetic rack until the solution is clear.
    9. Transfer the 50 μl supernatant to a clean PCR tube. Discard beads.
    10. To the 50 μl supernatant, add 50 μl (1.0X) of the resuspended AMPure XP beads and mix well on a vortex mixer or by pipetting up and down at least 10 times.
    11. Incubate for 5 minutes at room temperature.
    12. Quickly spin the tube in a microcentrifuge and place the tube on an appropriate magnetic rack to separate beads from the supernatant. After the solution is clear (about 5 minutes), discard the supernatant that contains unwanted fragments (Caution: do not discard the beads).
    13. Add 200 μl of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
    14. Repeat Step 14 once for a total of 2 washing steps.
    15. Briefly spin the tube, and put the tube back in the magnetic rack.
    16. Completely remove the residual ethanol, and air dry beads for 10 minutes while the tube is on the magnetic rack with the lid open.
    17. Elute DNA target from the beads with 25 μl nuclease-free water. Mix well on a vortex mixer or by pipetting up and down, and put the tube in the magnetic rack until the solution is clear.
    18. Without disturbing the bead pellet, transfer 20 μl of the supernatant to a clean PCR tube and proceed to PCR enrichment.
    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.