NEBNext^® Fast DNA Fragmentation & Library Prep Set for Ion Torrent^™

Overview

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Starting Material: 10 ng–1 μg of DNA.

Note: For use with the Ion Xpress™ Barcode Adapters 1-16 Kit, a minimum of 100 ng starting material is recommended. Lower amounts may cause adaptor concatamerization.

DNA quality and composition can affect fragmentation. Be sure to use the highest quality DNA.

Note: For GC Rich content, add 1 μl of MgCl₂ to the reaction. If fragmentation is insufficient, add additional 10 mM (1 μl) of MgCl₂ to reach desired fragment size. See Figure 1 on product page  for typical fragment size distribution.

Protocol

1.1. Mix the following components in a sterile microfuge tube on ice:
DNA   1–15.5 μl
NEBNext DNA Fragmentation Reaction Buffer   2 μl
Sterile H₂O   variable
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Total volume   18.5 μl

1.2. Vortex for 3 seconds, pulse spin and place on ice.

1.3. Vortex the vial of NEBNext DNA Fragmentation Master Mix for 3 seconds and pulse spin to collect liquid from the sides of the tube.

1.4. Add 1.5 μl of NEBNext DNA Fragmentation Master Mix to the microfuge tube, vortex for 3 seconds and pulse spin.

Note: For DNA with an AT content ≥ 70%, add 0.75 μl of NEBNext Fragmentation Master Mix and 0.75 μ l sterile H₂O.

1.5. Incubate in a thermal cycler for 20 minutes at 25°C, followed by 10 minutes at 70°C, hold at 4°C.

1.6. Pulse spin the microfuge tube and return to ice.

2. Preparation of Adaptor Ligated DNA

2.1. Add the following to the microfuge tube:

2.2. The total volume in the microfuge tube should be 40 μl. Mix the contents by pipetting up and down several times.

2.3. Incubate in a thermal cycler for 15 minutes at 25°C, followed by 5 minutes at 65°C, hold at 4°C.

2.4. Add 5 μl Stop Buffer, vortex and pulse-spin.

If performing bead based size selection, proceed directly to size selection using AMPure XP Beads. If using E-Gel or Agarose gel for size selection, proceed to Cleanup of Adaptor Ligated DNA before proceeding to gel based size selection.

3. Size Selection Using AMPure XP Beads

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 is calculated based on the original volume of DNA solution.

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 Table 1.1.

AMPure XP Bead-based Dual Bead Size Selection for 100 bp Inserts

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.

3.1. Add 55 μl 0.1X TE to the adaptor ligated DNA from Section 2, Step 2.4 to bring the total volume to 100 μl.

3.2. 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.

3.3. Incubate for 5 minutes at room temperature.

3.4. 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 3.4) to the beads. Unwanted smaller fragment sizes will not bind to the beads.

3.5. Add 15 μl (0.15X) resuspended AMPure XP Beads to the supernatant, mix well and incubate for 5 minutes at room temperature.

3.6. 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).

3.7. 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.

3.8. Repeat Step 3.7 once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.

3.9. Keeping the tube on the magnetic rack, with the cap open, air dry the beads for up to 5 minutes.

Caution: Do not overdry the beads. This may result in lower recovery of DNA target

3.10. Remove the tube from the magnet. Elute DNA target from beads into 42 μ l 0.1X TE. Mix well on a vortex mixer or by pipetting up and down, and incubate for 2 minutes at room temperature.

3.11. Put the tube in a magnetic rack until the solution is clear. Transfer approximately 40 μl of the supernatant to a clean tube.

3.12. Proceed to PCR Amplification of Adaptor Ligated DNA.

4. Cleanup of Adaptor Ligated DNA

4.1. Add 81 µl (1.8 x volume) of AMPure XP Beads to the sample and mix by pipetting up and down.

4.2. Incubate for 5 minutes at room temperature.

4.3. Pulse-spin the tube and place in a magnetic rack for approximately 2-3 minutes until the beads have collected to the wall of the tube and the solution is clear.

4.4. Carefully remove and discard the supernatant without disturbing the beads that contain the DNA target.

4.5. Keep the tube on the magnet and add 200 µl freshly prepared 80% ethanol. Incubate to room temp for 30 seconds and carefully remove and discard the supernatant.

4.6. Repeat Step 4.5 once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.

4.7. Keeping the tube in the magnetic rack, with the cap open, air dry the beads for 5 minutes at room temperature.

Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.

4.8. Remove the tube from the magnet. Resuspend the beads in 25 μl of 0.1X TE (volume may be adjusted for specific size selection protocol). Incubate for 2 minutes at room temperature.

4.9. Pulse spin the tube, and place in the magnetic rack until the beads have collected to the side of the tube and the solution is clear.

4.10. Transfer approximately 20 μl of the supernatant to a clean tube. Be careful not to transfer any beads.

4.11. Proceed to PCR Amplification of Adaptor Ligated DNA (Section 5).

5. PCR Amplification of Adaptor Ligated DNA

A precipitate can form upon thawing of the NEBNext Q5 Hot Start HiFi PCR Master Mix. To ensure optimal performance, place the master mix at room temperature while performing size selection/cleanup of adaptor-ligated DNA. Once thawed, gently mix by inverting the tube several times.

6. Cleanup of Amplified Library

6.1. Add 90 μl (0.9X volume) of AMPure XP Beads to the sample and mix by pipetting up and down.

6.2. Incubate for 5 minutes at room temperature.

6.3. Pulse spin the tube and place in a magnetic rack for 2–3 minutes until the beads have collected to the side of the tube and the solution is clear.

6.4. Carefully remove and discard the supernatant without disturbing the beads.

6.5. Keep the tube on the magnet and add 200 μl freshly prepared 80% ethanol. Incubate at room temperature, for 30 seconds, and carefully remove and discard the supernatant.

6.6. Repeat Steps 6.5 once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.

6.7. Keeping the tube in the magnetic rack, with the cap open, air dry the beads for 5 minutes.

Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.

6.8. Remove the tube from the magnet. Resuspend the beads in 25 μl of 0.1X TE. Mix well on a vortexer or by pipetting up and down, and incubate for 2 minutes at room temperature.

6.9. Put the tube on the magnetic rack until the solution is clear.

6.10. Transfer approximately 20 μl of supernatant to a fresh tube. Be careful not to transfer any beads.

6.11. Dilute 2-3 μl of the library 1:4 in 0.1X TE. Assess library quality on the Bioanalyzer.