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 .
