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  • Size Selection (E6285)

    Size distribution and library yields will vary depending on the size selection method. Size selection can be performed using a number of methods including E-Gel size select gels, standard 2% agarose gels, or AMPure XP Beads (see protocol below). For E-Gel size select gels, select adaptor ligated DNA in the 190–230 bp range for 100 bp libraries and 290–330 bp range for 200 bp read lengths. For AMPure XP bead size selection, use the protocol below. Expect size distributions in the range of 230–270 for 100 bp reads and 310–370 for 200 bp reads.

    Table 1: Recommended Conditions for Dual Bead-based Size Selection

    BEAD:DNA RATIO* INSERT SIZE (bp)
    100 bp 200 bp
    1st Bead Selection 0.9X 0.7X
    2nd Bead Selection 0.15X 0.15X
    *Bead:DNA ratio is calculated based on the original volume of DNA solution.

    AMPure XP Bead-based Dual Bead Size Selection for 100 bp Inserts
    Caution: The following protocol is for size selecting libraries with a 100 bp insert from a 100 μl volume. For libraries with a 200 bp insert, please use the bead:DNA ratio listed in the chart above.

    1st Bead Selection to Remove Large Fragments:
    This step is used to bind the large, unwanted fragments to the beads. The supernatant will contain the desired fragments
    1. Add 90 µl (0.9X) 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 on a magnetic rack to separate the beads from the supernatant. After the solution is clear (about 5 minutes), carefully transfer the supernatant to a new tube (Caution: do not discard the supernatant). Discard beads that contain the large fragments.

    2nd Bead Selection to Remove Small Fragments and to Bind DNA Target: 

    This step will bind the desired fragment sizes (contained in the supernatant from step 3) to the beads. Unwanted smaller fragment sizes will not bind to the beads.

    1. Add 15 µl (0.15X) resuspended AMPure XP beads to the supernatant, mix well and incubate for 5 minutes at room temperature.
    2. Put the tube on a 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 (Caution: do not discard beads).
    3. Add 500 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.
    4. Repeat Step 6 once.
    5. Keeping the tube on the magnetic rack, with the cap open, air dry the beads for 5 minutes.
    6. Elute DNA target from beads into 45 µl 0.1X TE. Mix well on a vortex mixer or by pipetting up and down, and put the tube in a magnetic rack until the solution is clear.
    7. Transfer approximately 40 µl of the supernatant to a clean tube. Be careful not to transfer any beads.
    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.