CTR:Notebook/PIRE/2015/02/02

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RAD Library Prep - Greenbuls and Sunbirds Plate 3

  • 96 well plate of 50ng of DNA 10uL

2015-01-31:

  • Digestion
    • Mix and add to each well:
      • 0.68 uL water
      • 1.20 uL NEBuffer 4
      • 0.12 uL SbfI-HF
    • Incubation (RADIGEST: Greenbuls on SORK, Sunbirds on TONY)
      • 37 degrees for 60 minutes
      • 65 degrees for 20 minutes
  • P1 adapter ligation
    • Add 2 uL indexed P1 adapter (10nM) to each well
    • Mix and add to each well:
      • 1.28 uL water
      • 0.40 uL NEBuffer 4
      • 0.16 uL ATP
      • 0.16 uL T4 Ligase
    • Incubation (RADLIG: Greenbuls on SORK, Sunbirds on TONY)
      • 20 degrees for 60 minutes
      • 65 degrees for 20 minutes

2015-02-01:

  • Clean up
    • Take 5 uL from each well and combine to 1.7 mL tube
    • AMPure bead clean up:
      • Use 430 uL beads to DNA (1:1)
      • 2 washes of 800 uL 80% EtOH
      • Elute in 100 uL LowTE
  • Sonication
    • BioRuptor NGS: Left - 8 cycles of 15 seconds on, 90 seconds off, High Power. Right - Additional 6 cycles for Greenbuls, 3 cycles for Sunbirds.

1) 2μL sheared Greenbul DNA, 2) 2uL 100bp low scale ladder, 3) 2uL sheared Sunbird DNA

2015-02-02:

  • Blunt End Repair
    • 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
    • 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
    • Add 16.5 uL water for a total of 100 uL
    • AMPure bead size selection:
      • 45 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
    • 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 Greenbul DNA template, 2) 5μL Greenbul PCR product, 3) 2 uL 100 bp low scale ladder, 4) 1μL Sunbird DNA template, 5) 5μL Sunbird PCR product
→Greenbul PCR does not look brighter than template, but may not be half as bright. Also, gel ran twice because first one failed, leaving only 40μL of PCR product left.

2015-02-03:

  • Re-run PCR amplification
    • 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)
    • PCR cycle (NEBPCR on JOHN Block B)


1) 1μL Greenbul DNA template, 2) 5μL Greenbul PCR product, 3) 2 uL 100 bp low scale ladder, 4) 1μL Sunbird DNA template, 5) 5μL Sunbird PCR product

Because Greenbuls did not seem to amplify well, proceeded with Sunbird library only:

  • Bead clean up
    • Use 45 uL AMPure beads (1:1)
    • 2 washes of 200 uL 80% EtOH
    • Elute in 30 uL LowTE
  • Quant DNA library using Qubit HS: 9.6 ng/μL
  • Run 1μL library + 2μL H2O on BioAnalyzer DNA High Sensitivity Chip (2015-02-06)

  • Dilute library to 10 nM in 10 uL and send to Berkeley for sequencing (Illumina HiSeq 100 SR) (2015-02-09)

Reprep of Greenbuls Plate 3 library

2/18/2015-2/23/2015

  • New enzyme and ligase used
  • Digestion and ligation cycles on TONY
  • First bead clean up: 440μL beads
  • Sonication: 8 cycles of 15 seconds on, 30 seconds off, High Power

2μL sheared DNA run on surface tension gel

  • Blunt End Repair, P2 ligation, and PCR cycles on JOHN Block B
  • Size selection: 45μL, 25μL beads
  • PCR using 5μL DNA

1) 2μL 100bp ladder, 2) 1μL template, 3) 5μL PCR product

  • Final bead clean up: 45μL beads
  • Qubit HS: 12.94 ng/μL
  • BioAnalyzer: 1μL library diluted to 5μL

  • Sent to Berkeley for seq: 10nm (2.01μL) in 10μL EBT buffer 2015-03-16




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