BestRAD Library Prep - COLO Plate 1 and COYE Plate 2
- 96 well plate of 100ng of DNA in 10uL volume
- Digestion (2017-06-14)
- Mix and add to each well:
- 0.68 uL water
- 1.20 uL NEBuffer 4
- 0.12 uL SbfI-HF
- Incubation:
- COLO P1, RAPDIG on TONY
- COLO P2, BEDIG on SORK
- 37 degrees for 60 minutes
- 80 degrees for 20 minutes
- BestRAD SbfI adapter ligation (2017-06-14)
- Add 2 uL annealed BestRAD SbfI adapters (50nM) to each well, using newly diluted plate of adaptors sent from UC Berkeley June 2016
- 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:
- COLO P1, RAPLIG on TONY
- COLO P2, BESTLIG on SORK
- 20 degrees for overnight (16 hours)
- 65 degrees for 20 minutes
- 1st Clean up (2017-06-15)
- 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 380 uL beads to DNA (1:1), per tube
- 2 washes of 800 uL 80% EtOH each
- Elute in 105 uL Low TE per tube
- Sonication (2017-06-15)
- 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 COLO P1 sheared DNA
2) 2uL 100bp low scale ladder,
3) 2uL COLO P2 sheared DNA
- Bind Ligated fragments to Dynabeads(2017-06-15)
- 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(2017-06-15)
- 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 (2017-06-19)
- 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 (2017-06-19)
- 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 (2017-06-19)
- 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 (2017-06-19)
- 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 (2017-06-19)
- 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 (BestRAD TestPCR on SORK Lab's Thermocycler):
- 98°C for 30 seconds
- 15 cycles of:
- 98°C for 10 seconds
- 65°C for 75 seconds
- 65°C for 5 minutes
1) 5uL COLO P1 PCR Product
2) 2uL 100bp low scale ladder,
3) 5uL COLO P2 PCR Product
- Final PCR amplification (2017-06-19)
- 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 (BestRAD FinalPCR on SORK Lab's Thermocycler):
- 98°C for 30 seconds
- 10 cycles of:
- 98°C for 10 seconds
- 65°C for 75 seconds
- 65°C for 5 minutes
1) 5uL COLO P1 PCR Product
2) 2uL 100bp low scale ladder,
3) 5uL COLO P2 PCR Product
- Size Selection Bead Clean Up (2017-06-22)
- 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: 2.2 ng/μL
- Plate 2: 2.04 ng/μL
- Run 1μL on BioAnalyzer DNA High Sensitivity Chip (2017-06-23)
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