NEBNext® Ultra™ DNA Library Prep Protocol for Illumina® With Size Selection (E7370)
Mix the following components in a sterile nuclease-free tube (Total volume 65 μl): End Prep Enzyme Mix   3.0 μl; End Repair Reaction Buffer (10X) 6.5 μl; Fragmented DNA   55.5 μl.
Mix by pipetting.
Quick spin to collect all liquid from the sides of the tube.
Place in a thermocycler, with the heated lid on, and run the following program:TimeTemperature30 minutes20°C30 minutes65°CHold4°C.
Add the following components directly to the End Prep reaction mixture and mix well (Total volume 83.5 μl): Blunt/TA Ligase Master Mix   15 μl; NEBNext Adaptor for Illumina   2.5 μl; Ligation Enhancer   1 μl.
Mix by pipetting.
Quick spin to collect all liquid from the sides of the tube.
Incubate at 20°C for 15 minutes in a thermal cycler.
Add 3 μl of USER™ enzyme to the ligation mixture from step 8.
Mix well and incubate at 37°C for 15 minutes.
Vortex AMPure XP beads to resuspend.
Add 13.5 μl dH2O to the ligation reaction for a 100 μl total volume.
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.
Incubate for 5 minutes at room temperature.
Quickly spin the tube.
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.
Add 25 μl resuspended AMPure XP beads to the supernatant.
Mix well and incubate for 5 minutes at room temperature.
Quickly spin the tube.
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).
Wash #1: Add 200 μl of 80% freshly prepared ethanol to the tube while in the magnetic stand.
Wash #1: Incubate at room temperature for 30 seconds.
Wash #1: Carefully remove and discard the supernatant.
Wash #2: Add 200 μl of 80% freshly prepared ethanol to the tube while in the magnetic stand.
Wash #2: Incubate at room temperature for 30 seconds.
Wash #2: Carefully remove and discard the supernatant.
Wash #3: Add 200 μl of 80% freshly prepared ethanol to the tube while in the magnetic stand.
Wash #3: Incubate at room temperature for 30 seconds.
Wash #3: Carefully remove and discard the supernatant.
Air the dry beads for 10 minutes while the tube is on the magnetic stand with the lid open.
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.
Mix the following components in sterile strip tubes (Total volume   50 μl): Adaptor Ligated DNA Fragments   23 μl; NEBNext High Fidelity 2X PCR Master Mix   25 μl; Index Primer   1 μl; Universal PCR Primer  1 μl.
PCR using the following cycling conditions: CYCLE STEPTEMPTIMECYCLESInitial Denaturation98°C30 seconds1Denaturation98°C10 seconds6-15*Annealing65°C30 secondsExtension72°C30 secondsFinal Extension72°C5 minutes1Hold4°C∞.
Vortex AMPure XP beads to resuspend.
Add 50 μl of resuspended AMPure XP beads to the PCR reactions (~ 50 μ l).
Mix well by pipetting up and down at least 10 times.
Incubate for 5 minutes at room temperature.
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).
Wash #1: Add 200 μl of 80% ethanol to the PCR plate while in the magnetic stand.
Wash #1:  Incubate at room temperature for 30 seconds.
Wash #1: Carefully remove and discard the supernatant.
Wash #2: Add 200 μl of 80% ethanol to the PCR plate while in the magnetic stand.
Wash #2:  Incubate at room temperature for 30 seconds.
Wash #2: Carefully remove and discard the supernatant.
Air dry the beads for 10 minutes while the PCR plate is on the magnetic stand with the lid open.
Elute DNA target from beads into 33 μl 10 mM Tris-HCl, pH 8.0 or 0.1X TE.
Mix well by pipetting up and down at least 10 times.
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 transfer 28 μl supernatant to a new PCR tube.
Dilute the library 5 fold with water, and check the size distribution on an Agilent high sensitivity chip.
