RAD Library Prep - Skinks Plate 3 (48 wells, Reruns)
- 48 wells with 100ng of DNA in 20uL
- Digestion (2015-08-25)
- Mix and add to each well:
- 1.36 uL water
- 2.40 uL CutSmart Buffer
- 0.24 uL SbfI-HF (new)
- Incubation (RADIGEST on TONY):
- 37 degrees for 60 minutes
- 65 degrees for 20 minutes
- P1 adapter ligation (2015-08-25)
- Add 4 uL indexed P1 adapter (10nM) to each well, using new plate of adapters made in-house
- Mix and add to each well:
- 2.56 uL water
- 0.8 uL NEBuffer 4
- 0.32 uL ATP
- 0.32 uL T4 Ligase (new)
- Incubation (RADLIG on TONY):
- 20 degrees for 60 minutes
- 65 degrees for 20 minutes
- Clean up (2015-08-26)
- Take 10 uL from each well and combine to 1.7 mL tube
- AMPure bead clean up:
- Used 430 uL beads to DNA (1:1)
- 2 washes of 800 uL 80% EtOH
- Elute in 100 uL LowTE
- Sonication - edited to get smaller average fragments (2015-08-26)
- BioRuptor NGS: 10 cycles of 30 seconds on, 90 seconds off, High Power
1) 2uL 100bp low scale ladder,
2) 2uL sheared DNA
- Additional 2 cycles of 30 seconds on, 90 seconds off, High Power
1) 2uL 100bp low scale ladder,
2) 2uL sheared DNA
- Blunt End Repair (2015-08-26)
- 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 (2015-08-26)
- 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 - edited to select for smaller insert (2015-08-26)
- Add 16.5 uL water for a total of 100 uL
- AMPure bead size selection:
- 55 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 (2015-08-26)
- 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
- Initially ran on 2% E-gel, but could not see library/template. Re-ran samples on 1% agarose gel.
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product
→Visible amplification, but size range difficult to discern.
- Re-run PCR to improve visibility and get more amplification (2015-08-27)
- 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)
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product
→Successful amplification.
- Clean up (2015-09-02, with Mice Plate 1)
- 45μL beads
- Added a second cleanup using ~28μL beads
- Ran gel to see final product
2μL Skink library on left
- Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2015-09-03)
- Sent 10nM to Berkeley for sequencing (Illumina HiSeq 100 PE) (2015-09-03)
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PIRE RAD Library Preps
