CTR:Notebook/PIRE/2015/04/01

PIRE RAD Library Preps

Main project page Previous entry      Next entry

RAD Library Prep - Skinks Plate 2 96 well plate of 50ng of DNA 10uL Digestion (2015-03-31) Mix and add to each well: 0.68 uL water 1.20 uL NEBuffer 4 0.12 uL SbfI-HF Incubation (RADIGEST on TONY): 37 degrees for 60 minutes 65 degrees for 20 minutes P1 adapter ligation (2015-03-31) Add 2 uL indexed P1 adapter (10nM) to each well, using new plate of adapters made in-house 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 (RADLIG on TONY): 20 degrees for 60 minutes 65 degrees for 20 minutes Clean up (2015-04-01) Take 5 uL from each well and combine to 1.7 mL tube AMPure bead clean up: Used 460 uL beads to DNA (1:1) 2 washes of 800 uL 80% EtOH Elute in 100 uL LowTE Sonication (2015-04-01) BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power 1) 2uL 100bp low scale ladder, 2) 2uL sheared DNA Additional 6 cycles of 15 seconds on, 90 seconds off, High Power 1) 2uL 100bp low scale ladder, 2) 2uL sheared DNA Blunt End Repair (2015-04-02) 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 (NEBENDRP on JOHN Block B): 20 degrees for 30 minutes 65 degrees for 30 minutes P2 adapter ligation (2015-04-02) 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 (2015-04-02) 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 (2015-04-02) 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 (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) 1 uL DNA template, 2) 5 uL PCR product, 3) 2 μL 100bp ladder Re-run PCR (2015-04-03) Mix: 10 uL DNA 25 uL NEBNext High Fidelity 2x PCR Master Mix 13 uL water 1 uL P1 adapter primer (25 uM) 1 uL P2 adapter primer (25 uM) PCR cycle (NEBPCR on JOHN Block B) 1) 5 uL PCR product, 2) 1 μL template, 3) 2 μL 100bp ladder Qubit: 5.98 ng/μL Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2015-02-06) RAD Library Re-prep - Skinks Plate 2 Reprep from plate. Digestion (2015-04-29) RADIGEST on TONY P1 adapter ligation (2015-04-29) Used new plate of adapters made in-house RADLIG on TONY Clean up (2015-04-30) Used 432μL Serapure beads to DNA (1:1) Sonication (2015-04-30) BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power 1) 2uL sheared DNA, 2) 2uL 100bp low scale ladder Blunt End Repair (2015-04-30) New NEBNext kit reagents used NEBENDRP on JOHN Block A P2 adapter ligation (2015-04-30) New NEBNext kit reagents used NEBLIGAT on JOHN Block A Size selection (2015-04-30) AMPure bead size selection: 45 uL beads to remove large fragments 25 uL beads to remove small fragments PCR amplification (2015-04-30) NEBPCR on JOHN Block A 1) 2uL 100bp low scale ladder, 2) 5uL of PCR product, 3) 1uL of template Qubit: 8.74 ng/uL 2nd PCR amplification (2015-05-01) Used 10 uL of template NEBPCR on JOHN Block A 1) 2uL 100bp low scale ladder, 2) 5uL of PCR product Bead clean up Use 45 uL AMPure beads (1:1) 2 washes of 200 uL 80% EtOH Elute in 30 uL LowTE Picogreen: 5.43 ng/uL Qubit: 9.82 ng/uL Ran 3μL on BioAnalyzer DNA High Sensitivity Chip (2015-02-06)