CTR:Notebook/PIRE/2015/08/31

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RAD Library Prep - 48-sample libraries for Butterflies, Frogs, Mice, Skinks

  • 48 wells with 100ng of DNA in 20uL: Butterflies Plate 1 + Frogs Plate 1, Mice Plate 1 + Skinks Plate 4
  • Digestion (2015-08-31)
    • Mix and add to each well:
      • 1.36 uL water
      • 2.40 uL CutSmart Buffer
      • 0.24 uL SbfI-HF (new)
    • Incubation (B+F: RADIGEST on SORK; M+S: RADIGEST on TONY):
      • 37 degrees for 60 minutes
      • 65 degrees for 20 minutes
  • P1 adapter ligation (2015-08-31)
    • Add 4 uL indexed P1 adapter (10nM) to each well, using new plate of adapters made in-house
    • Mix and add to each well:
      • 2.56 uL water
      • 0.8 uL CutSmart Buffer
      • 0.32 uL ATP
      • 0.32 uL T4 Ligase (new)
    • Incubation (B+F: RADLIG on SORK; M+S: RADLIG on TONY):
      • 20 degrees for 60 minutes
      • 65 degrees for 20 minutes
  • Clean up (2015-08-31)
    • Take 10 uL from each well and combine to 1.7 mL tube
    • AMPure bead clean up:
      • Used 440-460 uL beads to DNA (1:1)
      • 2 washes of 800 uL 80% EtOH
      • Elute in 100 uL LowTE
  • Sonication (2015-09-01)
    • BioRuptor NGS: 12 cycles of 30 seconds on, 90 seconds off, High Power


1) 2μL sheared Butterfly DNA
3) 2μL sheared Frog DNA
5) 2μL sheared Mouse DNA
7) 2μL sheared Skink DNA

  • Blunt End Repair (2015-09-01)
    • Mix:
      • 50.0 uL fragmented DNA
      • 5.5 uL water
      • 3.0 uL End Prep Enzyme Mix
      • 6.5 uL End Repair Reaction Buffer
    • Incubation (NEBENDRP on JOHN Block B):
      • 20 degrees for 30 minutes
      • 65 degrees for 30 minutes
  • P2 adapter ligation (2015-09-01)
    • Add to mix:
      • 15.0 uL Blunt/TA Ligase Master Mix
      • 2.5 uL P2 RAD adapter (5 uM)
      • 1.0 uL Ligation Enhancer
    • Incubation (NEBLIGAT on JOHN Block B):
      • 20 degrees for 15 minutes
  • Size selection - edited to select for smaller insert (2015-09-01)
    • Add 16.5 uL water for a total of 100 uL
    • AMPure bead size selection:
      • 55 uL beads to remove large fragments
      • 25 uL beads to remove small fragments
      • 3 washes of 200 uL 80% EtOH
      • Elute in 20 uL LowTE
  • PCR amplification (2015-09-01)
    • Mix:
      • 5 uL DNA
      • 25 uL NEBNext High Fidelity 2x PCR Master Mix
      • 18 uL water
      • 1 uL P1 adapter primer (25 uM)
      • 1 uL P2 adapter primer (25 uM)
    • PCR cycle (NEBPCR on JOHN Block B):
      • 98 degrees for 30 seconds
      • 15 cycles of:
        • 98 degrees for 10 seconds
        • 65 degrees for 30 seconds
        • 72 degrees for 30 seconds
      • 72 degrees for 5 minutes


  1. 1μL Butterfly template
  2. 5μL Butterfly PCR product
  3. 1μL Frog template
  4. 5μL Frog PCR product
  5. 2μL 100bp low scale ladder
  6. 1μL Mouse template
  7. 5μL Mouse PCR product
  8. 1μL Skink template
  9. 5μL Skink PCR product

→Amplification failed for Butterflies, Frogs, Skinks. Mouse shows some amplification.

  • Re-run Mouse PCR (2015-09-02)
    • Mix:
      • 10 uL DNA
      • 25 uL NEBNext High Fidelity 2x PCR Master Mix
      • 13 uL water
      • 1 uL P1 adapter primer (25 uM)
      • 1 uL P2 adapter primer (25 uM)

1) 2μL 100bp low scale ladder, 2) 1μL Mouse template, 3) 5μL Mouse PCR product
→ Clear amplification.

  • Bead clean up (2015-09-02, with Skinks plate 3)
    • Use 45μL AMPure beads (1:1)
    • Added second clean up using ~28μL beads (1:1)
    • Ran gel for final product

2μL Mouse library on right

  • PicoGreen: 1.54 ng/μL


  • Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2015-09-03)

  • Sent 10nM to Berkeley for sequencing (Illumina HiSeq 100 PE) (2015-09-03)



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