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  • Protocol (E6200) for Use With End User Supplied Primers and Adaptors

    Protocol

    Starting Material: 10 ng of chromatin-immunoprecipitated (ChIP) qPCR verified or control DNA, in ≤ 40 μl of water or elution buffer

    End Repair of ChIP DNA
    1. Dilute DNA Polymerase I, Large (Klenow) Fragment by mixing 1 μl of enzyme with 4 μl of sterile water in a fresh microfuge tube.
    2. In a sterile microfuge tube mix the following components:
      ChIP DNA 1–40 μl
      Phosphorylation Reaction Buffer (10X) 5 μl
      dNTP mix 2 μl
      T4 DNA Polymerase 1 μl
      Diluted DNA Pol I Klenow Fragment 1 μl
      T4 Polynucleotide Kinase 1 μl
      Sterile H2O variable
      Total volume 50 μl
    3. Incubate in a thermal cycler for 30 minutes at 20°C.
    Clean Up Using AMPure XP Beads
    1. Vortex beads to resuspend.
    2. Add 90 μl (1.8X) of resuspended AMPure XP beads to the reaction (~ 50 μl). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
    3. Incubate for 5 minutes at room temperature.
    4. 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.
    5. 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.
    6. Repeat Step 5 once.
    7. Air dry beads for 10 minutes while the tube/PCR plate is on the magnetic stand with the lid open.
    8. Elute DNA target from beads into 40 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.
    9. Transfer 34 μl of the supernatant to a clean LoBind tube, and store at –20°C.
    Alternatively, purify DNA sample on one purification column and elute in 34 μl of sterile dH2O or elution buffer.

    dA-Tailing of End Repaired DNA
    1. Mix the following components in a sterile microfuge tube:
      End Repaired DNA 34 μl
      NEBuffer 2 5   μl
      dATP 10 μl
      Klenow Fragment (3´→ 5´ exo) 1 μl
      Total volume 50 μl
    2. Incubate at 37°C for 30 minutes.
    Clean up Using AMPure XP Beads
    1. Vortex beads to resuspend.
    2. Add 90 μl (1.8X) of resuspended AMPure XP beads to the dA tailing reaction (~ 50 μl). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
    3. Incubate for 5 minutes at room temperature.
    4. 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.
    5. 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.
    6. Repeat Step 5 once.
    7. Air dry beads for 10 minutes while the tube/PCR plate is on the magnetic stand with the lid open.
    8. Elute DNA target from beads into 15 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.
    9. Transfer 10 μl of the supernatant to a clean LoBind tube, and store at –20°C.
    Alternatively, purify DNA sample on one purification column and elute in 10 μl of sterile dH2O or elution buffer.

    Adaptor Ligation of dA-Tailed DNA
    1. Mix the following components in a sterile microfuge tube:
      End Repaired, dA-Tailed DNA 10 μl
      Quick Ligation Reaction Buffer (2X) 15 μl
      1.5 µM DNA Adaptors*
      1 μl
      Quick T4 DNA Ligase 4 μl
      Total volume 30 μl
      * Adaptors are not included, use adaptors appropriate to specific application. If necessary adjust  the adaptor concentration to obtain a final adaptor to DNA molar ratio of 10:1.

    2. Incubate at 20°C for 15 minutes.

    Clean Up Using AMPure XP Beads
    1. Vortex AMPure XP beads to resuspend
    2. Add 54 μl (1.8X) of resuspended AMPure XP beads to the ligation reaction (~ 30 μl). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
    3. Incubate for 5 minutes at room temperature.
    4. 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.
    5. 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.
    6. Repeat Step 5 once.
    7. Air dry beads for 10 minutes while the tube/PCR plate is on the magnetic stand with the lid open.
    8. Elute DNA target from beads into 105 μl of dH2O. 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.
    9. Transfer 100 μl of the supernatant to a clean tube/PCR plate.
    Alternatively, purify DNA sample on one purification column and elute in appropriate volume of sterile dH2O or elution buffer for desired size selection.


    Size selection of Adaptor Ligated DNA Using AMPure XP Beads
    Note: (X) refers to original sample volume of 100 μl.
    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/PCR plate on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully trans- fer 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 care- fully 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 lid open.
    9. Elute DNA target from beads into 30 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.

    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.

        10.  Transfer 23 μl of the supernatant to a clean PCR tube and proceed to enrich- ment.

    Alternatively, size selection can be performed using a number of methods including E-Gel size select gels or standard 2% agarose gels. NEB's 100 bp ladder (NEB #N3231) can be used to determine the size of the fragments. Isolate library fragments in the 175–225 base pair range.

    Purify the DNA on one purification column and elute in 25 μl of sterile water or elution buffer.

    PCR Enrichment of Adaptor Ligated DNA
    1. Mix the following components in a sterile microfuge tube:
      Adaptor ligated DNA 23 μl
      NEBNext High-Fidelity 2X PCR Master Mix**  25 μl
      Primer 1* (25 μM) 1 μl
      Primer 2* (25 μM) 1 μl
      Total volume 50 μl
      * Primers are not included, use primers appropriate to specific application.
      **NEBNext High-Fidelity 2X PCR Master Mix will be replacing Phusion High-Fidelity PCR Master Mix. Both vials will be supplied for a limited time only.
    2. PCR cycling conditions
      Cycle step Temp Time Cycles
      Initial denaturation 98°C 30 sec 1
      Denaturation
      Annealing
      Extension
      98°C
      65°C
      72°C
      10 sec
      30 sec
      30 sec
      15
      Final extension 72°C
      4 °C
      5 min
      hold
      1
    Clean Up Using AMPure XP Beads
    1. Vortex beads to resuspend.
    2. 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.
    3. Incubate for 5 minutes at room temperature.
    4. 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.
    5. 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.
    6. Repeat Step 5 once.
    7. Air dry beads for 10 minutes while the tube/PCR plate is on the magnetic stand with the lid open.
    8. Elute DNA target from beads into 20 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.
    9. Transfer 15 μl of the supernatant to a clean LoBind tube, and store at –20°C.
    Alternatively, purify sample on one purification column and elute in 15 μl of sterile water or elution buffer.
    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 around 275 bp is expected.

    Figure 1: Bioanalyzer traces of final library.