CTR:Notebook/Migrant/2016/04/13: Difference between revisions

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**Combined both tubes of sheared DNA and ran on a gel
**Combined both tubes of sheared DNA and ran on a gel
[[Image:WIFLP1P2Sheared4152016.PNG|60px]]  
[[Image:WIFLP1P2Sheared4152016.PNG|60px]]  
1) 2uL WIFL P1 sheared DNA
1) 2uL YWAR P1 sheared DNA
2) 2uL 100bp low scale ladder,  
2) 2uL 100bp low scale ladder,  
3) 2uL WIFL P2 sheared DNA
3) 2uL YWAR P2 sheared DNA


* Bind Ligated fragments to Dynabeads(2016-04-15)
* Bind Ligated fragments to Dynabeads(2016-04-15)

Revision as of 14:54, 25 July 2016

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BestRAD Library Prep - YWAR Plate 1 & Plate 2

  • 96 well plates of 100ng of DNA in 10uL volume
  • Digestion (2016-04-13)
    • Mix and add to each well:
      • 0.68 uL water
      • 1.20 uL NEBuffer 4
      • 0.12 uL SbfI-HF
    • Incubation:

YWAR P1, RAPDIG on TONY YWAR P2, BEDIG on SORK

      • 37 degrees for 60 minutes
      • 80 degrees for 20 minutes
  • BestRAD SbfI adapter ligation (2016-04-13)
    • 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:

YWAR P1, RAPLIG on TONY YWAR P2, BESTLIG on SORK

      • 20 degrees for overnight (16 hours)
      • 65 degrees for 20 minutes
  • 1st Clean up (2016-04-15)
    • 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 343 uL beads to DNA (1:1), per tube
      • 2 washes of 800 uL 80% EtOH each
      • Elute in 105 uL LowTE per tube
  • Sonication (2016-04-15)
    • BioRuptor NGS: 10 cycles of 15 seconds on, 90 seconds off, High Power
    • Combined both tubes of sheared DNA and ran on a gel

1) 2uL YWAR P1 sheared DNA 2) 2uL 100bp low scale ladder, 3) 2uL YWAR P2 sheared DNA

  • Bind Ligated fragments to Dynabeads(2016-04-15)
    • 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(2016-04-15)
    • 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 B)
  • 2nd Clean up (2016-04-15)
    • 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
  • Blunt End Repair (2016-04-17)
    • 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 (2016-04-17)
    • 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 (2016-04-17)
    • 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 (2016-04-17)
    • 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

1) 2uL 100bp low scale ladder 2) 2uL YWAR P1 PCR product 3) 2uL YWAR P2 PCR product

  • Final PCR amplification (2016-04-17)
    • 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

1) 2uL 100bp low scale ladder 2) 2uL YWAR P1 PCR product 3) 2uL YWAR P2 PCR product

  • Size Selection Bead Clean Up (2016-04-17)
    • Add 55 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:
    • Plate 1: 32.86 ng/μL
    • Plate 2: 23.22 ng/μL
  • Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2016-04-17)
  • Plate 1:

  • Plate 2:
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