Protocol (E6040) for use with End User Supplied Primers and Adaptors

1. Vortex AMPure XP beads to resuspend.
2. Add 160 μl (1.6X) of resuspended AMPure XP beads to the ligation reaction. Mix thoroughly 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% 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.
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 by adding 50 μl sterile water to the beads. 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. Without disturbing the bead pellet, carefully transfer 42 μl of the supernatant to a fresh, sterile microfuge tube.

1. Mix the following components in a sterile microfuge tube:

   End Repaired DNA	42 μl
   NEBNext dA-Tailing Reaction Buffer (10X)	5   μl
   Klenow Fragment (3'→5' exo-)	3 μl
   Total volume	50 μl

2. Incubate at 37°C for 30 minutes.

1. Vortex AMPure XP beads to resuspend.
2. Add 90 μl (1.8X) of resuspended AMPure XP beads to the ligation reaction. Mix thoroughly 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 by adding 30 μl sterile water to the beads. 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. Without disturbing the bead pellet, carefully transfer 25 μl of the supernatant to a fresh, sterile microfuge tube.

1. Vortex AMPure XP beads to resuspend
2. Add 90 μl (1.8X) of resuspended AMPure XP beads to the ligation reaction (~53 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% 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.
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 targetby adding 105 μl sterile 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.
9. Transfer 100 μl of the supernatant (or desired volume) to a new tube/well, and proceed to bead based size selection.

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 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 25 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.

1. Mix the following components in a sterile microfuge tube:

   DNA	20 μl
   Primer 1 (25 μM)	2.5 μl
   Primer 2 (25 μM)	2.5 μl
   NEBNext High-Fidelity 2X PCR Master Mix*	25 μl
   Total volume	50 μl

   **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	4-8*
   Final extension	72°C 4 °C	5 min hold	1

*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.

1. Vortex AMPure XP 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 30 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 25 μl of the supernatant to a clean LoBind tube, and store at –20°C.
   Alternatively, adaptor ligated DNA can be purified on one purification column. Purify DNA on one QIAquick column and elute in 27 µl of 0.1X TE Buffer.
10. Dilute the library 20 fold with nuclease free water, and assess the library quality on a Bioanalyzer (high sensitivity chip). Check that the electropherogram shows a narrow distribution with a peak size approximately 300–320 bp