CTR:Notebook/PIRE/2014/07/09

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PIRE RAD Library Preps Main project page
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RAD Library Prep - Olive Sunbirds Plate 2, Little Greenbuls Plates 1 & 2

  • 96 well plate of 50ng of DNA 10uL


For Digestion & Ligation steps:

  • Sunbirds Plate 2 - 2014-07-09, Sork thermocycler
  • Greenbuls Plate 1 - 2014-07-11, Tony thermocycler
  • Greenbuls Plate 2 - 2014-07-14, Sork thermocycler


  • Digestion
    • Mix and add to each well:
      • 0.68 uL water
      • 1.20 uL NEBuffer 4
      • 0.12 uL SbfI-HF
    • Incubation:
      • 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:
      • 20 degrees for 60 minutes
      • 65 degrees for 20 minutes


The rest of the steps were done with all 3 libraries:

  • Clean up - 2014-07-15
    • Take 5 uL from each well and combine to 1.7 mL tube
    • AMPure bead clean up:
      • Use ~460 uL beads to DNA (1:1)
      • 2 washes of 800 uL 80% EtOH
      • Elute in 100 uL LowTE
  • Sonication - 2014-07-15
    • BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power

2μL of sheared DNA in each well
2014-07-16:

  • Blunt End Repair
    • Mix:
      • 50.0 uL fragmented DNA
      • 5.5 uL LowTE
      • 3.0 uL End Prep Enzyme Mix
      • 6.5 uL End Repair Reaction Buffer
    • Incubation: 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: 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: 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) 1 μL Sunbird Plate 2 template, 2) 5 μL Sunbird Plate 2 PCR product
3) 1 μL Greenbul Plate 1 template, 4) 5 μL Greenbul Plate 1 PCR product
5) 1 μL Greenbul Plate 2 template, 6) 5 μL Greenbul Plate 2 PCR product


Troubleshooting

  • Tried re-prep of leftover sheared DNA (Blunt End Repair → PCR), but no difference


  • Used old P1 primer in amplification


→ Works. Could mean that P1 adapter ligation failed and only fragments with P2 adapters on both ends are amplifying.


  • Start with leftover DNA from Greenbuls Plate 2 and re-shear to see if overshearing caused lack of P1 adapter to most fragments.

1) Pre-cleaned DNA (post-P1-ligation), 2) Cleaned DNA, 3) Sheared DNA

  • 1st shearing step looks good - proceed to Blunt End Repair → PCR

1) 1μL template, 2) 5μL PCR product
→ Still no amplification. Sonication is not the issue.


  • Test Sunbirds Plate 1 template and replicate PCR, and check if failing templates will amplify with only P2 Primer

1&2) Sunbirds Plate 1 template and PCR product, 3&4) Greenbuls Plate 2 old template and PCR product
→ PCR with good template still works. P2 primer only does not work at all

RAD Library Prep -



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