RAD Library Prep - 48-sample libraries for Butterflies, Frogs, Mice, Skinks
- 48 wells with 100ng of DNA in 20uL: Butterflies Plate 1 + Frogs Plate 1, Mice Plate 1 + Skinks Plate 4
- Digestion (2015-08-31)
- Mix and add to each well:
- 1.36 uL water
- 2.40 uL CutSmart Buffer
- 0.24 uL SbfI-HF (new)
- Incubation (B+F: RADIGEST on SORK; M+S: RADIGEST on TONY):
- 37 degrees for 60 minutes
- 65 degrees for 20 minutes
- P1 adapter ligation (2015-08-31)
- 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 CutSmart Buffer
- 0.32 uL ATP
- 0.32 uL T4 Ligase (new)
- Incubation (B+F: RADLIG on SORK; M+S: RADLIG on TONY):
- 20 degrees for 60 minutes
- 65 degrees for 20 minutes
- Clean up (2015-08-31)
- Take 10 uL from each well and combine to 1.7 mL tube
- AMPure bead clean up:
- Used 440-460 uL beads to DNA (1:1)
- 2 washes of 800 uL 80% EtOH
- Elute in 100 uL LowTE
- Sonication (2015-09-01)
- BioRuptor NGS: 12 cycles of 30 seconds on, 90 seconds off, High Power
1) 2μL sheared Butterfly DNA,
3) 2μL sheared Frog DNA,
5) 2μL sheared Mouse DNA,
7) 2μL sheared Skink DNA
- Blunt End Repair (2015-09-01)
- 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 (2015-09-01)
- 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 (2015-09-01)
- 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 (2015-09-01)
- 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
- 1μL Butterfly template
- 5μL Butterfly PCR product
- 1μL Frog template
- 5μL Frog PCR product
- 2μL 100bp low scale ladder
- 1μL Mouse template
- 5μL Mouse PCR product
- 1μL Skink template
- 5μL Skink PCR product
→Amplification failed for Butterflies, Frogs, Skinks. Mouse shows some amplification.
- Re-run Mouse PCR (2015-09-02)
- 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) 2μL 100bp low scale ladder, 2) 1μL Mouse template, 3) 5μL Mouse PCR product
→ Clear amplification.
- Bead cleanup (2015-09-02, with Skinks plate 3)
- Bead clean up
- Use 45 uL AMPure beads (1:1)
- 2 washes of 200 uL 80% EtOH
- Elute in 30 uL LowTE
- Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2015-09-03)
- Dilute library to 10 nM in 10 uL and send to Berkeley for sequencing (Illumina HiSeq 100 SR) (2015-02-09)
|