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  • Protocol for DNA (E7335)

    Introduction

    Starting Material: 1–5 μg of DNA fragmented to 200 bp.

    End Repair and dA-Tailing Steps
    1. Perform end repair and dA-Tailing according to the appropriate protocol.

    Protocol

    1. Adaptor Ligation of dA-Tailed DNA
      1. Mix the following components in a sterile microfuge tube:
        Reagent Set Protocol (NEB #E6000 )
        dA-Tailed DNA 10 μl
        Quick Ligation Reaction Buffer (2X) 25 μl
        Adaptor (15 μM) 10 μl
        Quick T4 DNA Ligase 5 μl
        --------------------------------------------------------------
        Total volume should be 50 μl

        Master Mix Protocol (NEB #E6040 )
        dA-Tailed DNA 25 μl
        Quick Ligation Reaction Buffer (5X) 10 μl
        Adaptor (15 μM) 10 μl
        Quick T4 DNA Ligase 5 μl
        --------------------------------------------------------------
        Total volume should be 50 μl
      2. Incubate in a thermal cycler for 15 minutes at 20°C.
      3. Add 3 μl of USER enzyme mix by pipetting up and down, and incubate at 37°C for 15 minutes.
      4. Vortex AMPure XP beads to resuspend.
      5. Add 90 μl of resuspended AMPure XP beads to the ligation reaction (~ 53 μl). Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times.
      6. Incubate for 5 minutes at room temperature.
      7. Put the tube/PCR plate on an appropriate magnetic stand 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.
      8. Add 200 μl of 80% freshly prepared ethanol to the tube/PCR plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
      9. Repeat Step 8 once.
      10. Air dry beads for 10 minutes while the tube/PCR plate is on the magnetic stand with the lid open.
      11. Elute DNA target by adding 102 μl water to the beads for bead-based size selection as detailed in the next section, or at desired volume for size selection using E-Gel size select gels or standard 2% agarose gels. Mix well on a vortex mixer or by pipetting up and down, and put the tube/PCR plate in the magnetic stand until the solution is clear.
      12. Transfer 100 μl of supernatant (or desired volume) to a new tube/well, and proceed to bead-based size selection.
    2. Alternately, adaptor ligated DNA can be purified on one purification column. Elute in sterile H2O in volume desired for subsequent size selection.

      Size Select Adaptor Ligated DNA Using AMPure XP Beads.

      Caution: The following size selection protocol is for libraries with 200 bp inserts only. For libraries with larger fragment inserts, please optimize bead:DNA ratio accordingly.
      1. Add 80 μl (0.8X) resuspended AMPure XP beads to 100 μl DNA solution. 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. Place the tube/PCR plate on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully transfer the supernatant to a new tube/well (Caution: do not discard the supernatant). Discard beads that contain the large fragments.
      4. Add 20 μl (0.2X) resuspended AMPure XP beads to the supernatant, mix well and incubate for 5 minutes at room temperature.
      5. Put the tube/PCR plate on an appropriate magnetic stand 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 (Caution: do not discard beads).
      6. Add 200 μl of 80% freshly prepared ethanol to the tube/PCR plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
      7. Repeat Step 6 once.
      8. Air dry beads for 10 minutes while the tube/PCR plate is on the magnetic stand with the lid open.
      9. Elute DNA target from beads into 22 μl water or 0.1X TE buffer. Mix well on a vortex mixer or by pipetting up and down, and put the tube/PCR plate in the magnetic stand until the solution is clear.
      10. Transfer 20 μl of the supernatant to a clean PCR tube and proceed to enrichment.
      Alternatively, adaptor ligated DNA can be size selected using a number of other methods including E-Gel size select gels or standard 2% agarose gels. Purify DNA sample on one column and elute in 22 μl of sterile water or elution buffer.


    3. PCR Enrichment of Adaptor Ligated DNA
      1. Mix the following components in a sterile microfuge tube:

        DNA 20 μl
        Universal PCR Primer (25 μM) 2.5 μl
        Index Primer (X)* (25 μM) 2.5 μl
        Phusion® High Fidelity PCR Master Mix with HF Buffer (2X) 25 μl
        ---------------------------------------------------------------------
        Total volume 50 μl
        *Note: The NEBNext Multiplex Oligos for Illumina (Index Primers 1–12) contains 1–12 PCR Primers, each with a different index. For each reaction, only one of the 12 PCR primer indices is used during the PCR step.
      2. PCR cycling conditions:
        CYCLE STEP TEMP TIME CYCLES
        Initial Denaturation 98°C 30 seconds 1
        Denaturation
        Annealing
        Extension
        98°C
        65°C
        72°C
        10 seconds
        30 seconds
        30 seconds
        4–8*
        Final Extension 72°C 5 minutes 1
        Hold 4°C
        *If library construction was performed with 5 μg of starting material, use 4 cycles of amplification. If starting material was 1 μg, use 6–8 cycles of amplification. However, optimization of PCR cycle number may be required to avoid over-amplification.
      3. Vortex AMPure XP beads to resuspend.
      4. Add 50 μl (1X) of resuspended AMPure XP beads to the PCR reactions (~ 50 μl). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
      5. Incubate for 5 minutes at room temperature.
      6. Put the tube/PCR plate on an appropriate magnetic stand 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.
      7. Add 200 μl of 80% ethanol to the tube/PCR plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
      8. Repeat Step 7 once
      9. Air dry beads for 10 minutes while the tube/PCR plate is on the magnetic stand with the lid open.
      10. Elute DNA target from beads into 27 μl of 0.1X TE. Mix well on a vortex mixer or by pipetting up and down, and put the tube/PCR plate in the magnetic stand until the solution is clear.
      11. Transfer 25 μl of the supernatant to a clean LoBind tube, and store at -20°C.
        Alternately, adaptor ligated DNA can be purified on one purification column. Elute in 30 µl of 0.1X TE Buffer.
      12. Dilute the library 20 fold with nuclease free water, and assess the library quality on a Bioanalyzer (Agilent high sensitivity chip). Check that the electropherogram shows a narrow distribution with a peak size approximately 300–320 bp.