CTR:Notebook/PIRE/2016/03/01

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BestRAD Library Prep - Butterflies Plate 2

  • 96 well plate of 50ng of DNA 10uL
  • Digestion (2016-02-29)
    • Mix and add to each well:
      • 0.68 uL water
      • 1.20 uL NEBuffer 4
      • 0.12 uL SbfI-HF
    • Incubation (RAPDIG on TONY):
      • 37 degrees for 60 minutes
      • 80 degrees for 20 minutes
  • BestRAD SbfI adapter ligation (2016-02-29)
    • Add 2 uL annealed BestRAD SbfI adapters (50nM) to each well, using new plate of adapters sent from UC Berkeley
    • 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 (RAPLIG on TONY):
      • 20 degrees for overnight (18 hours)
      • 65 degrees for 20 minutes
  • 1st Clean up (2016-03-01)
    • Take 8 uL from each well and combine to 1.7 mL tube, split entire volume into 2 separate tubes
    • AMPure bead clean up:
      • Used 350 uL beads to DNA (1:1), per tube
      • 2 washes of 800 uL 80% EtOH each
      • Elute in 105 uL LowTE per tube
  • Sonication (2016-03-01)
    • BioRuptor NGS: 10 cycles of 15 seconds on, 90 seconds off, High Power
    • Combined both tubes of sheared DNA and ran on a gel

Shearedbestrad03012016.jpg 1) 2uL 100bp low scale ladder, 2) 2uL sheared DNA

  • Bind Ligated fragments to Dynabeads(2016-03-02)
    • Transfer 200 μL of prepared Dynabeads to the tube of sonicated DNA (~200 μL)
    • 3 washes of 150 μL 1X B+W Buffer
    • 2 washes with 150 μL 56°C 1X B+W Buffer
  • Liberate DNA from Dynabeads(2016-03-02)
    • 2 washes of 100ul 1X NEB Buffer 4
    • Resuspend beads in 40 μL of 1X NEB Buffer 4
    • Add 2 μL of SbfI-HF
    • Incubate tube at 37°C for 60 min.(LIBDNA on JOHN, Block A)
  • 2nd Clean up (2016-03-02)
    • Take 45 uL of supernatant
    • AMPure bead clean up:
      • Used 45 uL beads to DNA (1:1)
      • 2 washes of 200 uL 80% EtOH
      • Elute in 56 uL LowTE
  • Image after second clean up:

BestRad Dynabeadcleanup 02252016.jpg 1) 2 μL 100bp ladder, 2) 2 μL DNA after clean up

  • Blunt End Repair (2016-03-03)
    • Mix:
      • 55.5 uL fragmented DNA
      • 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
  • NEBNext adapter ligation (2016-03-03)
    • Add to mix:
      • 15.0 uL Blunt/TA Ligase Master Mix
      • 2.5 uL NEBNext adaptor for Illumina (1.5 uM)
      • 1.0 uL Ligation Enhancer
    • Incubation (NEBLIGAT on JOHN Block B):
      • 20°C for 15 minutes
    • Added 3 μL of USER enzyme to ligation mixture.
    • Incubation (USERENZ on JOHN Block B)
      • 37°C for 15 minutes
  • Size selection (2016-03-03)
    • 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
  • First Test PCR amplification (2016-03-03)
    • Mix:
      • 5 uL DNA
      • 25 uL NEBNext Q5 Hot Start HiFi PCR Master Mix
      • 10 uL H2Owater
      • 5 uL Index 1 Primer (10 uM)
      • 5 uL Universal PCR Primer (10 uM)
    • PCR cycle (NEBTESTP on JOHN Block B):
      • 98°C for 30 seconds
      • 15 cycles of:
        • 98°C for 10 seconds
        • 65°C for 75 seconds
      • 65°C for 5 minutes

BestRadTestPCR03032016.jpg 1) 2 μL 100bp ladder, 2) 5 μL PCR product

  • Final PCR amplification (2016-03-03)
    • Mix:
      • 15 uL DNA
      • 25 uL NEBNext Q5 Hot Start HiFi PCR Master Mix
      • 5 uL Index 1 Primer (10 uM)
      • 5 uL Universal PCR Primer (10 uM)
    • PCR cycle (NEBFINPC on JOHN Block B):
      • 98°C for 30 seconds
      • 12 cycles of:
        • 98°C for 10 seconds
        • 65°C for 75 seconds
      • 65°C for 5 minutes

BestRadFinalPCR03032016 B.jpg 1) 2 μL 100bp ladder, 2) 5 μL PCR product

  • Bead clean up (2016-03-04)
    • Use 45 uL AMPure beads (1:1)
    • 2 washes of 200 uL 80% EtOH
    • Elute in 30 uL LowTE
  • Qubit: 4.46 ng/μL
  • Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2016-03-04)

BestRad ButterflyP2 Bioanalyzer.png

  • Bioanalyzer Results
  • Fragment range is too wide, second size selection using Ampure XP beads is done to get rid of adaptor primers (~120bp) and the larger fragments (>500bp).
  • Second Size Selection (2016-03-08)
    • Add 72 uL low TE 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 30 uL LowTE
  • Qubit: 2.26 ng/μL
  • Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2016-03-09)

BestRad 03082016.png