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 PIRE RAD Library Preps
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RAD Library Prep - Olive Sunbirds Plate 2, Little Greenbuls Plates 1 & 2
- 96 well plate of 50ng of DNA 10uL
For Digestion & Ligation steps:
- Sunbirds Plate 2 - 2014-07-09, Sork thermocycler
- Greenbuls Plate 1 - 2014-07-11, Tony thermocycler
- Greenbuls Plate 2 - 2014-07-14, Sork thermocycler
- Digestion
- Mix and add to each well:
- 0.68 uL water
- 1.20 uL NEBuffer 4
- 0.12 uL SbfI-HF
- Incubation:
- 37 degrees for 60 minutes
- 65 degrees for 20 minutes
- P1 adapter ligation
- Add 2 uL indexed P1 adapter (10nM) to each well
- 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:
- 20 degrees for 60 minutes
- 65 degrees for 20 minutes
The rest of the steps were done with all 3 libraries:
- Clean up - 2014-07-15
- Take 5 uL from each well and combine to 1.7 mL tube
- AMPure bead clean up:
- Use ~460 uL beads to DNA (1:1)
- 2 washes of 800 uL 80% EtOH
- Elute in 100 uL LowTE
- Sonication - 2014-07-15
- BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power
2μL of sheared DNA in each well
2014-07-16:
- Blunt End Repair
- Mix:
- 50.0 uL fragmented DNA
- 5.5 uL LowTE
- 3.0 uL End Prep Enzyme Mix
- 6.5 uL End Repair Reaction Buffer
- Incubation: on John Block B
- 20 degrees for 30 minutes
- 65 degrees for 30 minutes
- P2 adapter ligation
- 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: on John Block B
- 20 degrees for 15 minutes
- Size selection
- 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
- 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: 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 μL Sunbird Plate 2 template, 2) 5 μL Sunbird Plate 2 PCR product
3) 1 μL Greenbul Plate 1 template, 4) 5 μL Greenbul Plate 1 PCR product
5) 1 μL Greenbul Plate 2 template, 6) 5 μL Greenbul Plate 2 PCR product
→ PCR products do not look 2x as bright as the template. No significant amplication.
Troubleshooting
- Tried re-prep of leftover sheared DNA (Blunt End Repair → PCR), but no difference
- Used old P1 primer in amplification
→ Works. Could mean that P1 adapter ligation failed and only fragments with P2 adapters on both ends are amplifying.
- Start with leftover DNA from Greenbuls Plate 2 and re-shear to see if overshearing caused lack of P1 adapter to most fragments.
1) Pre-cleaned DNA (post-P1-ligation), 2) Cleaned DNA, 3) Sheared DNA
- 1st shearing step looks good - proceed to Blunt End Repair → PCR
1) 1μL template, 2) 5μL PCR product
→ Still no amplification. Sonication is not the issue.
- Test Sunbirds Plate 1 template and replicate PCR, and check if failing templates will amplify with only P2 Primer
1&2) Sunbirds Plate 1 template and PCR product, 3&4) Greenbuls Plate 2 old template and PCR product
→ PCR with good template still works. P2 primer only does not work at all
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PIRE RAD Library Preps
