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  • NEBNext Fill-in and ssDNA Isolation Module Protocol (E6070, E6071)

    Introduction

    Recommended: 
    Removal of small fragments using Agencourt AMPure Beads or gel size selection.

    Protocol

    1. Transfer 50 μl of Hydrophilic Streptavidin Magnetic Beads to a 1.5 ml tube.

    2. Using a magnet, pellet the beads and remove the buffer.

    3. Wash beads twice with 100 μl of 2X Bead Binding Buffer, pelleting the beads with a magnet to remove the buffer after each wash.

    4. Resuspend beads in 25 μl of 2X Bead Binding Buffer.

    5. Add 25 μl adapter-ligated DNA fragments to the beads.

    6. Vortex and place on a tube rotator at room temperature for 20 minutes.

    7. Using a magnet, wash the beads twice with 100 μl (1X) Bead Wash Buffer, pelleting the beads with a magnet to remove the buffer after each wash.

    8. Mix the following components in a separate sterile microfuge tube:
      Molecular Biology Grade Water: 42 μl
      Adapter Fill-in Reaction Buffer: 5 μl
      Bst DNA Polymerase, Large Fragment: 3 μl

    9. Transfer the 50 μl Fill-in Reaction Mix to the beads.

    10. Vortex lightly and incubate at 37°C for 20 minutes.

    11. Wash beads twice with 100 μl of 1X Bead Wash Buffer, pelleting the beads with a magnet to remove the buffer after each wash.

    12. Prepare Melt Solution:
      10 N NaOH: 125 μl
      Water: 9.875 ml

    13. Prepare Neutralization Solution in a 1.5 ml tube.
      3 M sodium acetate, pH 5.2: 10 μl
      Column binding buffer with pH indicator: 500 μl

    14. Add 50 μl of Melt Solution to the beads.

    15. Vortex well, pellet the beads with a magnet.

    16. Carefully transfer the Melt Solution containing the ssDNA Fragment Library to 1.5 ml tube containing the Neutralization Solution.

    17. Repeat steps 14–16, adding the second round of Melt Solution containing the ssDNA Fragment Library to the same 1.5 ml tube containing the Neutralization Solution and the first round of Melt Solution and ssDNA Fragments. Adjust pH if necessary by adding an additional 5 μl of 3 M sodium acetate.

    18. Purify DNA on one column without adding any additional column binding buffer. Wash column twice with column wash buffer to remove all residual salts. Elute in 25 μl of sterile dH2O or elution buffer.