BestRAD Library Prep - PABU Plate 1
- 96 well plate of 75ng of DNA in 10uL volume
- Digestion (2016-04-18)
- 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 (2016-04-18)
- 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 (15 hours)
- 65 degrees for 20 minutes
- 1st Clean up (2016-04-19)
- 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 335 uL beads to DNA (1:1), per tube
- 2 washes of 800 uL 80% EtOH each
- Elute in 105 uL LowTE per tube
- Sonication (2016-04-19)
- 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 100bp low scale ladder,
2) 2uL sheared DNA
- Bind Ligated fragments to Dynabeads (2016-04-19)
- 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 (2016-04-19)
- 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 (2016-04-19)
- 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 (2016-04-19)
- 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 (2016-04-19)
- 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):
- Added 3 μL of USER enzyme to ligation mixture.
- Incubation (USERENZ on JOHN Block B)
- Size selection (2016-04-20)
- 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 (2016-04-20)
- 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) 5ul PCR product
- Final PCR amplification (2016-04-20)
- 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
- 11 cycles of:
- 98°C for 10 seconds
- 65°C for 75 seconds
- 65°C for 5 minutes
1) 2uL 100bp low scale ladder
2) 5ul PCR product
- Size Selection Bead Clean Up (2016-04-20)
- 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
- Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2016-06-01)
- Sent 10nM to UC Davis for sequencing (Illumina HiSeq 4000 (PE 100)) (2016-06-07)
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