NEBNext FFPE DNA Repair Mix (M6630) - Protocol for use with NEBNext Ultra DNA Library Prep Kit for Illumina (E7370) and NEBNext High-Fidelity 2X PCR Master Mix (E7375)

Symbols
This caution sign signifies a step in the protocol that has multiple paths
leading to the same end point but is dependent on a user variable, like the
amount of input DNA.
Colored bullets indicate the cap color of the reagent to be added to a reaction.

Starting Material: (5 ng–1 μg) of fragmented FFPE DNA

2.1 NEBNext FFPE Repair
  1. Mix the following components in a sterile nuclease-free tube:
    FFPE DNA 53.5 μl
     (green) FFPE DNA Repair Buffer 6.5 μl
     (green) NEBNext FFPE DNA Repair Mix 2 μl
    --------------------------------------------------------------------------------------------
    Total volume 62 μl
  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.
2.2 Cleanup Using AMPure XP Beads

  1. Vortex AMPure XP Beads to resuspend.
  2. Add 186 μl (3X) of resuspended AMPure XP Beads to the repair 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 the 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 5 minutes while the tube/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 by adding 60 μl 0.1X TE to the beads. Mix well on a vortex mixer or by pipetting up and down, and incubate for 2 minutes at room temperature. Put the tube/PCR plate in the magnetic stand until the solution is clear.
  9. Without disturbing the bead pellet, carefully transfer 55.5 μl of the supernatant to a fresh, sterile microfuge tube.
2.3 NEBNext End Prep
  1. Add the following directly to the FFPE repair reaction mixture:
    (green) End Repair Reaction Buffer (10X) 6.5 μl
    (green) End Prep Enzyme Mix 3.0 μl
    Repaired DNA from above (Step 9) 55.5 μl
    --------------------------------------------------------------------------------------------
    Total volume 65 μl
  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 set to 75°C, and run the follwing program:
    30 minutes @ 20°C
    30 minutes @ 65°C
    Hold at 4°C
2.4 Adaptor Ligation

 If DNA input is < 100 ng, dilute the NEBNext Adaptor for Illumina (provided at 15 μM) 10 fold in 10 mM Tris-HCl pH 7.5 with 10 mM NaCl to a final concentration of 1.5 μM; use immediately.

  1. Add the following components directly to the End Prep reaction mixture:
    (red) Blunt/TA Ligase Master Mix 15 μl
    (red) NEBNext Adaptor for Illumina (15 μM)* 2.5 μl
    (red) Ligation Enhancer 1 μl
    End Prep DNA mixture from above (Section 2.3) 65 μl
    --------------------------------------------------------------------------------------------
    Total volume 83.5 μl
    *The NEBNext adaptor is provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7600) Oligos for Illumina.
  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.
  4. Add 3 μl of (red) USER™ Enzyme to the ligation mixture from Step 3. Mix well and incubate at 37°C for 15 minutes.
    Note: This step is for use with NEBNext adaptors only. USER Enzyme can be found in the NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7600) Oligos for Illumina.

    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 cleanup of adaptor-ligated DNA. Once thawed, gently mix by inverting the tube several times.
2.5 Cleanup of Adaptor-ligated DNA

  1. Vortex AMPure XP Beads to resuspend
  2. Add 86.5 μl resuspended AMPure XP Beads to the ligation reaction. 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 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.
  6. Repeat Step 5 once.
  7. Air the dry 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.
  8. Remove the tube/plate from the magnet. Elute the DNA target from the beads by adding 22 μl of 10 mM Tris-HCl, pH 8.0 or 0.1X TE.
  9. Mix well by pipetting up and down, or on a vortex mixer.
  10. Quickly spin the tube and incubate for 2 minutes at room temperature.
  11. Place the tube on a magnetic stand. After the solution is clear (about 5 minutes), transfer 20 μl to a new PCR tube for amplification.
    Note: Be sure not to transfer any beads. Trace amounts of bead carryover may affect the optimal performance of the polymerase used in the NEBNext High-Fidelity 2X PCR Master Mix in the subsequent PCR step.
  12. Proceed to PCR Amplification.

2.6 PCR Amplification

Note: NEBNext Singleplex and Multiplex Oligos for Illumina (NEB #E7350, #E7335 and #E7500) now have new primer concentrations (10 μM). Please check oligo kit lot numbers to determine how to set up your PCR reaction.

Follow Section 2.6A if you are using the following oligos (10 μMprimer):
NEBNext Singleplex Oligos for Illumina (NEB #E7350) lot 0071412
NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335) lot 0091412
NEBNext Multiplex Oligos for Illumina (Set 2, NEB #E7500) lot 0071412
NEBNext Multiplex Oligos for Illumina (Dual Index Primers, NEB #E7600) all lots

Follow Section 2.6B if you are using the following oligos (25 μM primer):
NEBNext Singleplex Oligos for Illumina (NEB #E7350) lots 0051402 or 0061410
NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335) lots 0071402 or 0081407
NEBNext Multiplex Oligos for Illumina (Set 2, NEB #E7500) lots 0051402 or 0061407

2.6A PCR Amplification

  1. Mix the following components in sterile strip tubes:
    Adaptor Ligated DNA Fragments (from Step 11, Section 2.5) 20 μl
    (blue) Index Primer/i7 Primer*,** 2.5 μl
    (blue) Universal PCR Primer/i5 Primer*,*** 2.5 μl
    (blue) NEBNext High-Fidelity 2X PCR Master Mix 25 μl
    --------------------------------------------------------------------------------------------
    Total volume 50 μl
    * The primers are provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7600) Oligos for Illumina. For use with Dual Index Primers (NEB #E7600), look at the NEB #E7600 manual for valid barcode combinations and tips for setting up the PCR reaction.
    ** For use with NEBNext Multiplex Oligos (NEB #E7335 or #E7500) use only one Index Primer per PCR reaction. For use with Dual Index Primers (NEB #E7600) use only one i7 Primer per reaction.
    *** For use with Dual Index Primers (NEB #E7600) use only one i5 Primer per reaction.
  2. PCR cycling conditions:
    CYCLE STEP TEMP TIME CYCLES
    Initial Denaturation 98°C 30 seconds 1
    Denaturation
    Annealing
    Extension
    98°C
    65°C
    72°C
    10 seconds
    30 seconds
    30 seconds
    4–15*
    Final Extension 72°C 5 minutes 1
    Hold 4°C  
    *We suggest 4-6 cycles for 1 μg DNA input, 8-9 cycles for 100 ng, 11-12 cycles for 20 ng, and 13-15 cycles for 5 ng. Further optimization of PCR cycle number may be required.
  3. Proceed to Cleanup of PCR Amplification (Section 2.7).

2.6B PCR Amplification

  1. Mix the following components in sterile strip tubes:
    Adaptor Ligated DNA Fragments (from Step 11, Section 2.5) 20 μl
    (blue) Index Primer*,** 1 μl
    (blue) Universal PCR Primer* 1 μl
    (blue) NEBNext High-Fidelity 2X PCR Master Mix 25 μl
    Sterile H2O 3 μl
    --------------------------------------------------------------------------------------------
    Total volume 50 μl
    * The primers are provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335 or #E7500) Oligos for Illumina.
    ** For use with NEBNext Multiplex Oligos (NEB #E7335 or #E7500) use only one Index Primer per PCR reaction.
  2. PCR cycling conditions:
    CYCLE STEP TEMP TIME CYCLES
    Initial Denaturation 98°C 30 seconds 1
    Denaturation
    Annealing
    Extension
    98°C
    65°C
    72 °C
    10 seconds
    30 seconds
    30 seconds
    4–15*
    Final Extension 72°C 5 minutes 1
    Hold 4°C  
    *We suggest 4-6 cycles for 1 μg DNA input, 8-9 cycles for 100 ng, 11-12 cycles for 20 ng, and 13-15 cycles for 5 ng. Further optimization of PCR cycle number may be required.

2.7 Cleanup of PCR Amplification

  1. Vortex AMPure XP Beads to resuspend.
  2. Add 50 μl of resuspended AMPure XP Beads to the PCR reactions (~ 50 μl). 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 μl 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 μl of 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. Libraries can be stored at –20°C.
  9. Dilute 2–3 μl of the library 5 fold with 10 mM Tris or 0.1X TE and check the size distribution on an Agilent Bioanalyzer® (high sensitivity chip).
  10. A sharp peak at 128 bp corresponds to adaptor-dimer. We recommend repeating steps 1–9 in Section 1.7 if this occurs.
Example of a library prepared with normal human liver FFPE DNA.