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  • Purify the Double-stranded cDNA Using 1.8X Agencourt AMPure XP Beads

    For use with NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (E7530) and NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (E7420).



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     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.

    Protocol

    1. Vortex AMPure XP beads to resuspend.

    2. Add 144 μl (1.8X) of resuspended AMPure XP beads to the second strand synthesis reaction (~80 μl). Mix well on a vortex mixer or by pipetting up and down at least 10 times.

    3. Incubate for 5 minutes at room temperature.

    4. Quickly spin the tube in a microcentrifuge to collect any sample on the sides of the tube. Place the tube on an appropriate 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.

    5. 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.

    6. Repeat Step 5 once for a total of 2 washing steps.

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

    8. Elute the DNA target from the beads into 60 μl nuclease-free water. Mix well on a vortex mixer or by pipetting up and down. Quickly spin the tube and then place it in the magnetic rack until the solution is clear.

    9. Remove 55.5 μl of the supernatant and transfer to a clean nuclease-free PCR tube. 
     Note: If you need to stop at this point in the protocol samples can be stored at –20°C.