CTR:Notebook/Migrant/2017/06/08

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BestRAD Library Prep - COLO Plate 1 and COYE Plate 2 96 well plate of 100ng of DNA in 10uL volume Used SbfI instead of SbFI-HF by accident... Digestion (2017-06-07) Mix and add to each well: 0.68 uL water 1.20 uL NEBuffer 4 0.12 uL SbfI 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 (2017-06-07) 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 (2017-06-08) 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 (2017-06-08) 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(2017-06-08) 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(2017-06-08) 2 washes of 100ul 1X NEB Buffer 4 Resuspend beads in 40 μL of 1X NEB Buffer 4 Add 2 μL of SbfI Incubate tube at 37°C for 60 min.(LIBDNA on JOHN, Block B) 2nd Clean up (2017-06-08) 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 (2017-06-12) 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 (2017-06-12) 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 (2017-06-12) 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 (2017-06-12) 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 (2017-06-13) 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 12 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 (2017-06-13) 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: 0.87 ng/μL Plate 2: 1.65 ng/μL PCR failed, SbfI might be the issue...