BestRAD Library Prep - Butterflies Plate 2
- 96 well plate of 50ng of DNA 10uL
- Digestion (2016-02-24)
- 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 (2016-02-24)
- 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 (20 hours)
- 65 degrees for 20 minutes
- 1st Clean up (2016-02-25)
- Take 8 uL from each well and combine to 1.7 mL tube
- AMPure bead clean up:
- Used 710 uL beads to DNA (1:1)
- 2 washes of 800 uL 80% EtOH
- Elute in 100 uL LowTE
- Sonication (2016-02-25)
- BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power
1) 2uL 100bp low scale ladder,
2) 2uL sheared DNA
- BioRuptor NGS: Additional 2 cycles of 15 seconds on, 90 seconds off, High Power
1) 2uL 100bp low scale ladder,
2) 2uL sheared DNA
- Bind Ligated fragments to Dynabeads(2016-02-25)
- Transfer 200 μL of prepared Dynabeads to the tube of sonicated DNA
- 3 washes of 150 μL 1X B+W Buffer
- 2 washes with 150 μL 56°C 1X B+W Buffer
- Liberate DNA from Dynabeads(2016-02-25)
- 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 (2016-02-25)
- 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:
1) 2 μL 100bp ladder, 2) 2 μL DNA after clean up
- Blunt End Repair (2016-02-26)
- 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
- P2 adapter ligation (2016-02-26)
- 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 (2016-02-26)
- 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 (2016-02-26)
- Mix:
- 4 uL DNA
- 25 uL NEBNext High Fidelity 2x PCR Master Mix
- 19 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) 2 μL 100bp ladder, 2) 5 μL PCR product
- Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2015-02-06)
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PIRE RAD Library Preps
