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NEBNext_Protocol_RB.md

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##Protocol for use with NEBNext Ultra DNA Library Prep Kit for Illumina (E7370)

Starting material is RAD library prep, 55.5ul

1.1 NEBNext End Prep

  1. Mix the following in a sterile tube:

    Color Component Volume
    Green End Prep Enzyme Mix 3.0 ul
    Green End Repair Reaction Buffer (10X) 6.5 ul
    na Fragmented DNA 55.5 ul

    Total volume: 65 ul

  2. Mix by pipetting followed by a quick spin to collect all liquid from the sides of the tube.

  3. Place in a thermocycler, with the heated lid on, and run the follwing program: "NEB_DNA" -> "End Prep"

    Time Temp
    30 minutes 20°C
    30 minutes 65°C
    hold 4°C

1.2 Adaptor ligation

Because DNA input is < 100 ng, dilute the NEBNext Adaptor for Illumina (provided at 15 uM) 10-fold in 10 mM Tris-HCl to a final concentration of 1.5 uM, use immediately. ie, 1 ul adaptor into 9ul tris

  1. Add the following components directly to the End Prep reaction mixture and mix well:

    Color Component Volume
    red Blunt-TA Ligase Master Mix 15 ul
    red NEBNext Adaptor for Illumina* 2.5 ul
    red ligation enhancer 1ul

    Total volume 83.5 ul
    *the NebNext Adaptor for Illumina is in the Multiplex kit

  2. Mix by pipetting followed by a quick spin to collect all liquid from the sides of the tube.

  3. Incubate at 20°C for 15 minutes in a thermal cycler.

    • Protocol: "NEB_DNA" ->"Adaptor"

  4. Add 3 ul of USER enzyme to the ligation mixture from Step 3. (found in the multiplex kit)

  5. Mix well and incubate at 37°C for 15 minutes.

    • in thermocycler

###1.3 Size Selection of Adaptor-ligated DNA

The following size selection protocol is for libraries with 250-300 bp inserts only and ~400 bp average library size. For libraries with different size fragment inserts, refer to Table 1.1 for the appropriate volume of beads to be added. The size selection protocol is based on a starting volume of 100 ul.

put Q5 hot start at room temp to thaw

  1. Vortex AMPure XP Beads to resuspend.
  2. Add 13.5 ul dH2O to the ligation reaction for a 100 ul total volume.
  3. Add 42.5 ul of resuspended AMPure XP Beads to the 100 ul ligation reaction. Mix well by pipetting up and down at least 10 times.
  4. Incubate for 5 minutes at room temperature.
  5. Quickly spin the tube and 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.
  6. Add 22.5 ul resuspended AMPure XP Beads to the supernatant, mix well and incubate for 5 minutes at room temperature.
  7. Quickly spin the tube and 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).
  8. Add 200 ul 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.
  9. Repeat Step 8 once.
  10. Air dry the beads for 5 minutes while the tube is on the magnetic stand with the lid open. Caution: Do not overdry the beads. This may result in lower recovery of DNA target.
  11. Remove the tube/plate from the magnet. Elute the DNA target from the beads into 22 ul of 10 mM Tris-HCI or 0.1 X TE. Mix well on a vortex mixer or by pipetting up and down. Incubate for 2 minutes at room temperature.
  12. Quickly spin the tube and place it on a magnetic stand. After the solution is clear (about 5 minutes), transfer 15 ul to a new PCR tube for amplification.
  13. Proceed to PCR Amplification in Section 1.4.

1.4 PCR Enrichment of Adaptor Ligated DNA

First use 5ul DNA with 14 cycles. Check for dna conc, then use the other 17 ul with the appropriate amount of cycles.

  1. Mix the following components in a sterile nuclease-free tube:

    Color Component Volume
    blue NEBNext Q5 Hot Start HiFi PCR Master Mix 25 ul
    blue Index Primer 5 ul
    blue Universal PCR Primer 5 ul

  2. PCR Cycling Conditions:

  3. Proceed to Cleanup of PCR Amplification Section 1.5.

1.5 Cleanup of PCR Amplification

  1. Vortex AMPure XP Beads to resuspend.
  2. Add 45 ul of resuspended AMPure XP Beads to the PCR reactions (~ 50 ul). Mix well by pipetting up and down at least 10 times.
  3. Incubate for 5 minutes at room temperature.
  4. 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).
  5. Add 200 ul of 80% ethanol to the 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 the beads for 5 minutes while the PCR plate is on the magnetic stand with the lid open. Caution: Do not overdry the beads. This may result in lower recovery of DNA target.
  8. Remove the tube/plate from the magnet. Elute DNA target from beads into 33 ul 0.1X TE. Mix well by pipetting up and down at least 10 times. Quickly spin the tube and incubate at room temperature for 2 minutes.
  9. Place the sample on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully transfer ~30 ul supernatant to a new PCR tube. Libraries can be stored at –20°C.
  10. Check the size distribution on an Agilent Bioanalyzer® (high sensitivity chip).