CTR:Notebook/PIRE/Entry Base

From OpenWetWare

Jump to: navigation, search
PIRE RAD Library Preps Main project page

RAD Library Prep - Olive Sunbirds Plate 1

  • 96 well plate of 50ng of DNA 10uL
  • Digestion (2014-04-30)
    • 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 (2014-04-30)
    • 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
  • Clean up (2014-04-30)
    • Take 5 uL from each well and combine to 1.7 mL tube
    • AMPure bead clean up:
      • Use 480 uL beads to DNA (1:1)
      • 2 washes of 800 uL 80% EtOH
      • Elute in 100 uL LowTE
  • Sonication (2014-05-01)
    • BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power

1) 1uL pre-sheared DNA, 2) 2uL 100bp low scale ladder, 3) 3uL sheared DNA

  • Blunt End Repair (2014-05-01)
    • 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:
      • 20 degrees for 30 minutes
      • 65 degrees for 30 minutes
  • P2 adapter ligation (2014-05-01)
    • 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:
      • 20 degrees for 15 minutes
  • Size selection (2014-05-01)
    • 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 (2014-05-01)
    • 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:
      • 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 uL DNA template, 2) 2 uL 100 bp low scale ladder, 3) 5 uL PCR product



Personal tools