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  • Size Selection of Adaptor-ligated DNA (E7370)

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     This caution sign signifies a step in the protocol that has multiple paths leading to the same end point but is dependent on a user variable, like the amount of input DNA.

     Size selection is optional. If the starting material is less than 50 ng, size selection is not recommended. If you are not performing size selection, proceed to page 5 and perform clean up step prior to PCR amplification. The following size selection protocol is for libraries with 200 bp inserts only. For libraries with different size fragment inserts, refer to Table 1 for the appropriate volume of beads to be added. The size selection protocol is based on a starting volume of 100 μl.

    Protocol

    1. Vortex AMPure XP beads to resuspend.
    2. Add 13.5 μl dH2O to the ligation reaction for a 100 μl total volume.
    3. Add 55 μl of resuspended AMPure XP beads to the 100 μl ligation reaction. Mix well by pipetting up and down at least 10 times.
    4. Incubate for 5 minutes at room temperature.
    5. Quickly spin the tube and place the tube on an appropriate magnetic stand to separate the beads from the supernatant. After the solution is clear (about 5 minutes), carefully transfer the supernatant containing your DNA to a new tube(Caution: do not discard the supernatant). Discard the beads that contain the unwanted large fragments.
    6. Add 25 μl resuspended AMPure XP beads to the supernatant, mix well and incubate for 5 minutes at room temperature.
    7. Quickly spin the tube and place it on an appropriate magnetic stand to separate the beads from the supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant that contains unwanted DNA. Be careful not to disturb the beads that contain the desired DNA targets (Caution: do not discard beads).
    8. Add 200 μl of 80% freshly prepared ethanol to the tube while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
    9. Repeat Step 8 twice for a total of three washes.
    10. Air the dry beads for 10 minutes while the tube is on the magnetic stand with the lid open.
    11. Elute the DNA target from the beads into 28 μl of 10 mM Tris-HCl or 0.1 X TE, pH 8.0. Mix well on a vortex mixer or by pipetting up and down. Quickly spin the tube and place it on a magnetic stand. After the solution is clear (about 5 minutes), transfer 23 μl to a new PCR tube for amplification.
    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.

    Alternatively, Cleanup of Adaptor-ligated DNA without Size Selection
    1. Vortex AMPure XP beads to resuspend.
    2. Add 86.5 μl resuspended AMPure XP beads to the ligation reaction. Mix well by pipetting up and down at least 10 times. Vortex AMPure XP beads to resuspend.
    3. Incubate for 5 minutes at room temperature.
    4. Quickly spin the tube and place it 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).
    5. Add 200 μl of 80% freshly prepared ethanol to the tube while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
    6. Repeat Step 5 twice, for a total of three washes.
    7. Air the dry beads for 10 minutes while the tube is on the magnetic stand with the lid open.
    8. Elute the DNA target from the beads by adding 28 μl of 10 mM Tris-HCl, pH 8.0 or 0.1X TE.
      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.
    9. Mix well by pipetting up and down, or on a vortex mixer.
    10. Quickly spin the tube and place it on the magnetic stand.
    11. After the solution is clear (about 5 minutes), transfer 23ml a new PCR tube for amplification.
    Table 1: Recommended conditions for bead based size selection.
    LIBRARY
    PARAMETERS
    APPROXIMATE
    INSERT SIZE
    150 bp 200 bp 250 bp 300-400 bp 400-500 bp 500-700 bp

    Total Library Size (insert + adaptor)

    270 bp

    320 bp

    400 bp

    400-500 bp

    500-600 bp

    600-800 bp

    VOLUME TO
    BE ADDED (μl)
    1st Bead Selection 65 55 45 40 35 30
    2nd Bead Selection 25 25 25 20 15 15