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  • Clean Up of Amplified Library (E6270)

    Protocol

    1. Add 90 µl (0.9X volume) of AMPure XP Reagent to the sample and mix by pipetting up and down.

    2. Incubate for 5 minutes at room temperature.

    3. Pulse-spin the tube and place in a magnetic rack for approximately 3 minutes until the beads have collected to the wall of the tube and the solution is clear.

    4. Carefully remove and discard the supernatant without disturbing the beads.

    5. Keep the tube on the magnet and add 200 µl freshly prepared 80% ethanol. Incubate 30 seconds, and carefully remove and discard the supernatant.

    6. Repeat step 5.

    7. Keeping the tube in the magnetic rack, with the cap open, air dry the beads for 5 minutes at room temperature.
      Caution: Do not overdry the beads. This may result in lower recovery of DNA target.

    8. Remove the tube from the magnet. Resuspend the beads in 35 μl of 0.1X TE and incubate for 2 minutes at room temperature.

    9. Pulse-spin the tube, return to the magnet until the beads have collected to the wall of the tube and solution is clear. 

    10. Transfer approximately 30 µl of supernatant to a fresh tube. Be careful not to transfer any beads.

    11. Dilute 2-3 μl of the library 1:4 in 0.1X TE. Assess the library quality on a Bioanalyzer.

      Figure 1.1: Relative size distribution of Fragmented End Repaired DNA as seen using the Bioanalyzer® 2100 (Agilent Technologies, Inc.).
      1 μg of E. coli DNA was fragmented and end repaired for 20 minutes at 25°C, followed by 10 minutes at 70°C.
      Figure 1.2: Final Library Size distribution using E-Gel Size Selection.
      Figure 1.3: Final Library Size distribution using AMPure XP Beads.