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 PIRE RAD Library Preps
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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
- Bead clean up (2014-05-02)
- Use 45 uL AMPure beads (1:1)
- 2 washes of 200 uL 80% EtOH
- Elute in 30 uL LowTE
- Quant DNA library using PicoGreen and run (<3 ng/uL) on BioAnalyzer DNA High Sensitivity Chip
- Dilute library to 10 nM in 10 uL and send to Berkeley for sequencing (Illumina HiSeq 100 SR)
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PIRE RAD Library Preps
