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| <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> |
| ==RAD Library Prep - Olive Sunbirds Plate 1== | | ==RAD P1 Adapter Ordering== |
| * 96 well plate of 50ng of DNA 10uL
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| * Digestion (2014-04-30)
| | Adapter Sequences (sequences from Mike Miller's files)<br> |
| **Mix and add to each well:
| | Ordered in plate format from IDT (1 plate of top oligos, 1 plate of bottom oligos)<br> |
| ***0.68 uL water | | Parameters: |
| ***1.20 uL NEBuffer 4 | | *Scale: 100 nmole |
| ***0.12 uL SbfI-HF | | *Purification: Standard desalting |
| **Incubation: | | *Ship option: Wet |
| ***37 degrees for 60 minutes | | *Amount per well: Full yield |
| ***65 degrees for 20 minutes | | *Concentration: 50 μM |
| | *Dilutant: IDTE Buffer pH 8.0 |
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| * P1 adapter ligation (2014-04-30)
| | ==RAD P1 Adapter Annealing and Dilution== |
| **Add 2 uL indexed P1 adapter (10nM) to each well
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| **Mix and add to each well:
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| ***1.28 uL water
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| ***0.40 uL NEBuffer 4
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| ***0.16 uL ATP
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| ***0.16 uL T4 Ligase
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| **Incubation:
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| *** 20 degrees for 60 minutes
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| *** 65 degrees for 20 minutes
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| | |
| * Clean up (2014-04-30)
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| **Take 5 uL from each well and combine to 1.7 mL tube
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| **AMPure bead clean up:
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| ***Use 480 uL beads to DNA (1:1)
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| ***2 washes of 800 uL 80% EtOH
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| ***Elute in 100 uL LowTE
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| | |
| * Sonication (2014-05-01)
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| **BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power
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| [[Image:Sunbird1 sheared 2014-05-01.JPG|200px]]
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| 1) 1uL pre-sheared DNA,
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| 2) 2uL 100bp low scale ladder,
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| 3) 3uL sheared DNA
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| | |
| *Blunt End Repair (2014-05-01)
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| **Mix:
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| ***50.0 uL fragmented DNA
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| ***5.5 uL water
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| ***3.0 uL End Prep Enzyme Mix
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| ***6.5 uL End Repair Reaction Buffer
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| **Incubation:
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| ***20 degrees for 30 minutes
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| ***65 degrees for 30 minutes
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| | |
| *P2 adapter ligation (2014-05-01)
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| **Add to mix:
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| ***15.0 uL Blunt/TA Ligase Master Mix
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| ***2.5 uL P2 RAD adapter (5 uM)
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| ***1.0 uL Ligation Enhancer
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| **Incubation:
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| ***20 degrees for 15 minutes
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| | |
| *Size selection (2014-05-01)
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| **Add 16.5 uL water for a total of 100 uL
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| **AMPure bead size selection:
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| ***45 uL beads to remove large fragments
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| ***25 uL beads to remove small fragments
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| ***3 washes of 200 uL 80% EtOH
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| ***Elute in 20 uL LowTE
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| | |
| *PCR amplification (2014-05-01)
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| **Mix:
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| ***5 uL DNA
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| ***25 uL NEBNext High Fidelity 2x PCR Master Mix
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| ***18 uL water
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| *** 1 uL P1 adapter primer (25 uM)
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| *** 1 uL P2 adapter primer (25 uM)
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| **PCR cycle:
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| ***98 degrees for 30 seconds
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| ***15 cycles of:
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| ****98 degrees for 10 seconds
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| ****65 degrees for 30 seconds
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| ****72 degrees for 30 seconds
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| ***72 degrees for 5 minutes
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| [[Image:Sunbirds1 PCR 2014-05-01.JPG|200px]]
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| 1) 1 uL DNA template, 2) 2 uL 100 bp low scale ladder, 3) 5 uL PCR product
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| | |
| *Bead clean up (2014-05-02)
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| **Use 45 uL AMPure beads (1:1)
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| **2 washes of 200 uL 80% EtOH
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| **Elute in 30 uL LowTE
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| | |
| *Quant DNA library using PicoGreen and run (<3 ng/uL) on BioAnalyzer DNA High Sensitivity Chip
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| [[Image:Sunbirds1 bioanalyzer.jpg|450px]]
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| | |
| *Dilute library to 10 nM in 10 uL and send to Berkeley for sequencing (Illumina HiSeq 100 SR) (2014-05-05)
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| ==RAD Library Prep - == | | ==RAD Library Prep - == |
PIRE RAD Library Preps
