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3RAD Protocol

1. Restriction Digest and Adapter Ligation

Note: ligation must be performed immediately following restriction digest.

Restriction Digest

Reagents
  • 20,000 U/mL New England BioLabs Restriction Enzymes
  • 10X New England BioLabs CutSmart Buffer
  • 2.5 µM Read 1 and Read 2 3RAD Adapters
Final Concentrations

In 15 µL:

  • 20-100 ng Sample DNA
  • 1X Cutsmart Buffer
  • 10 U of read 1 restriction enzyme
  • 10 U of read 2 restriction enzyme
  • 10 U of adapter dimer restriction enzyme (Optional)
  • 0.33 µM read 1 adapter
  • 0.33 µM read 2 adapter
Procedure
Enzyme Digestion Master Mix 1X 116X
10X CutSmart Buffer 1.5 µL 174 µL
H2O 3.0 µL 348 µL
Enzyme 1 20 U/µL 0.5 µL 58 µL
Enzyme 2 20 U/µL 0.5 µL 58 µL
Enzyme 3 20 U/µL 0.5 µL 58 µL
Total volume 6 µL 696 µL

If not using third enzymes add 0.5 µL more of water per sample.

  1. Pipette 5 µL of DNA at a concentrations of 4-20 ng/µL onto a 96 well plate.
  2. Prepare enzyme digestion master mix.
  3. Pipette 50 µL of master mix into each well of a 12 well strip tube or 75 µ into each well of an 8 well strip tube.
  4. Using a multichannel pipette, add 6 µL master mix to each sample.
  5. Add 2 µL of each 2.5 µM adapter.
  6. Incubate samples:
    • No heated lid
    • 37 °C for 1 hour

Ligation

Reagents
  • 400,000 U/mL New England BioLabs T4 DNA Ligase
  • 10X New England Biolabs Ligase Buffer
  • Promega 10 mM rATP
Final Concentrations

In 20 µL:

  • 100 U of T4 DNA ligase
  • 0.25X ligase buffer
  • 0.75 mM rATP
Procedure
Ligation Master Mix 1X 106X
H2O 2.75 µL 291.5 µL
10X Ligase Buffer 0.5 µL 53 µL
10 mM rATP 1.5 µL 159 µL
Ligase 400,000 ∪/mL 0.25 µL 26.5 µL
Total volume 5 µL 530 µL
  1. Prepare ligation master mix.
  2. Pipette 43 µL of master mix into each well of a 12 well strip tube or 65 µL into each well of an 8 well strip tube.
  3. Using a multichannel pipette, add 5 µL ligation master mix to each sample.
  4. Incubate samples:
    • No heated lid
    • 2 cycles of:
      • 22 °C for 20:00
      • 37 °C for 10:00
    • 80 °C for 20:00

2. Bead Cleanup

  1. Pool 5 µL of each sample into a single 1.5 mL tube.
  2. Mix pooled sample with 1X Speedbeads and briefly vortex.
  3. Allow sample to sit for 1 minute.
  4. Place sample on magnet stand until solution is completely clear and magnets have been drawn to the side of the tube.
  5. Carefully pipette solution from tube and discard.
  6. Wash sample twice with 500 µL of 70% ETOH:
    1. Add 500 µL of 70% ETOH to tube and let sit for 1 minute.
    2. Carefully pipette ETOH from tube.
    3. Repeat once.
    4. Use toothpick to remove remaining drops of ETOH and let stand until no ETOH remains.
  7. Re-suspend in 25 µL of TLE Buffer Solution.

3. One Cycle PCR With iTru5-8N Primer

Reagents
  • Kapa HiFI PCR Kit
  • 5 µM iTru5 Primer
Final Concentrations

In 50 µL:

  • 5 µL of pooled DNA samples
  • 1X KAPA HiFi Fidelity Buffer
  • 0.3 µM iTru5 Primer
  • 0.3 mM each dNTP
  • 1 U of KAPA HiFi DNA Polymerase
Procedure
PCR Master Mix 1X 5X
H2O 29.5 µL 147.5 µL
5X Kapa HiFi Buffer 10 µL 50 µL
5 µM iTru5-8N Primer 3 µL 15 µL
10mM dNTP 1.5 µL 7.5 µL
KAPA Hifi Polymerase 1.0 µL 5 µL
Total volume 45 µL 225 µL
  1. Prepare PCR master mix.
  2. Add 45 µL master mix to 4 0.2 mL PCR tubes
  3. Add 5 µL pooled DNA to each PCR tubes
  4. Incubate samples at:
    • 98 °C for 2:00
    • 60 °C for 0:30
    • 72 °C for 5:00

4. Bead Cleanup

  1. Pool iTru5-8N PCR product into single 1.5 mL tube.
  2. Mix pooled sample with 1.5X Speedbeads and briefly vortex.
  3. Allow sample to sit for 1 minute.
  4. Place sample on magnet stand until solution is completely clear and magnets have been drawn to the side of the tube.
  5. Carefully pipette solution from tube and discard.
  6. Wash sample twice with 500 µL of 70% ETOH:
    1. Add 500 µL of 70% ETOH to tube and let sit for 1 minute.
    2. Carefully pipette ETOH from tube.
    3. Repeat once.
    4. Use toothpick to remove remaining drops of ETOH and let stand until no ETOH remains.
  7. Re-suspend in 25 µL TLE Buffer Solution.

5. Two Primer Amplification With P5 & iTru7 Primers

Reagents
  • Kapa HiFI PCR Kit
  • 5 µM iTru7 Primer
  • 5 µM P5 Primer
Final Concentrations

In 50 µL:

  • 5 µL of pooled DNA samples
  • 1X KAPA HiFi Fidelity Buffer
  • 0.3 µM iTru7 Primer
  • 0.3 µM P5 Primer
  • 0.3 mM each dNTP
  • 1 U of KAPA HiFi DNA Polymerase
Procedure
PCR Master Mix 1X 5X
H2O 26.5 µL 132.5 µL
5X Kapa HiFi Buffer 10 µL 50 µL
5 µM iTru7 Primer 3 µL 15 µL
5 µM P5 Primer 3 µL 15 µL
10mM dNTP 1.5 µL 7.5 µL
KAPA Hifi Polymerase 1.0 µL 5 µL
Total volume 45 µL 225 µL
  1. Prepare PCR master mix.
  2. Add 45 µL master mix to six 0.2 mL PCR tubes
  3. Add 5 µL pooled DNA to each of the six PCR tubes
  4. Incubate samples at:
    • 95 °C for 3:00
    • 6 cycles of:
      • 98 °C for 0:20
      • 60 °C for 0:15
      • 72 °C for 0:30
    • 72 °C 5:00

6. Bead Cleanup

  1. Pool P5-iTru7 PCR products into single 1.5 mL tube.
  2. Mix pooled sample with 2X Speedbeads and briefly vortex.
  3. Allow sample to sit for 1 minute.
  4. Place sample on magnet stand until solution is completely clear and magnets have been drawn to the side of the tube.
  5. Carefully pipette solution from tube and discard.
  6. Wash sample twice with 500 µL of 70% ETOH:
    1. Add 500 µL of 70% ETOH to tube and let sit for 1 minute.
    2. Carefully pipette ETOH from tube.
    3. Repeat once.
    4. Use toothpick to remove remaining drops of ETOH and let stand until no ETOH remains.
  7. Re-suspend in 35 µL TLE Buffer Solution.

7. Size Selection

Procedure

Size select DNA with BluePippin.

8. Final Amplification with P5 and P7 Primers

Reagents
  • Kapa HiFI PCR Kit
  • 5 µM P5 Primer
  • 5 µM P7 Primer
Final Concentrations

In 50 µL:

  • 5 µL of pooled DNA samples
  • 1X KAPA HiFi Fidelity Buffer
  • 0.3 µM P5 Primer
  • 0.3 µM P7 Primer
  • 0.3 mM each dNTP
  • 1 U of KAPA HiFi DNA Polymerase
Procedure
PCR Master Mix 1X 5X
H2O 26.5 µL 132.5 µL
5X Kapa HiFi Buffer 10 µL 50 µL
5 µM P5 Primer 3 µL 15 µL
5 µM P7 Primer 3 µL 15 µL
10mM dNTP 1.5 µL 7.5 µL
KAPA Hifi Polymerase 1.0 µL 5 µL
Total volume 45 µL 225 µL
  1. Prepare PCR master mix.
  2. Add 45 µL master mix to six 0.2 mL PCR tubes.
  3. Add 5 µL pooled DNA to each of the six PCR tubes.
  4. Incubate samples at:
    • 95 °C for 3:00
    • 12 cycles of:
      • 98 °C for 0:20
      • 60 °C for 0:15
      • 72 °C for 0:45
    • 72 °C 5:00

9. Bead Cleanup

  1. Pool P5-P7 PCR products into single tube.
  2. Mix pooled sample with 1X Speedbeads and briefly vortex.
  3. Allow sample to sit for 1 minute.
  4. Place sample on magnet stand until solution is completely clear and magnets have been drawn to the side of the tube.
  5. Carefully remove pipette solution from tube.
  6. Wash sample twice with 500 µL of 70% ETOH:
    1. Add 500 µL of 70% ETOH to tube and let sit for 1 minute.
    2. Carefully pipette ETOH from tube.
    3. Repeat once.
    4. Use toothpick to remove remaining drops of ETOH and let stand until no ETOH remains.
  7. Re-suspend in 25 µL 10mM Tris-HCl Buffer Solution.