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| Line 6: |
Line 6: |
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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== | | ==Frog and Mouse DNA QC== |
| * 96 well plate of 50ng of DNA 10uL
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|
| |
|
| * Digestion (2014-04-30)
| | Nov. 2014<br><br> |
| **Mix and add to each well:
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| ***0.68 uL water
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| ***1.20 uL NEBuffer 4
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| ***0.12 uL SbfI-HF
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| **Incubation:
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| ***37 degrees for 60 minutes
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| ***65 degrees for 20 minutes
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|
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| * P1 adapter ligation (2014-04-30)
| | [http://openwetware.org/images/f/f0/Frog_gDNA_Aliquots_SL.xlsx Frog Quant Results] |
| **Add 2 uL indexed P1 adapter (10nM) to each well
| | <br> |
| **Mix and add to each well:
| | Frog extracts from 2013. When run on PicoGreen/Qubit, concentrations were far lower than reported Nanodrop scores. Re-running Nanodrop at UCLA yielded similar numbers to reported concentrations. 1μL from 4 samples run on gel showed no bands.<br> |
| ***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)
| | [http://openwetware.org/images/7/7d/Mice-gDNA_SL.xlsx Mice Quant Results]<br> |
| **Take 5 uL from each well and combine to 1.7 mL tube
| | Mice extracts from 2013. PicoGreen showed higher concentrations than the frog extracts. 2μL of each extract run on gel to check for quality. |
| **AMPure bead clean up:
| |
| ***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
| |
| **Incubation:
| |
| ***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
| |
| ***2.5 uL P2 RAD adapter (5 uM)
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| ***1.0 uL Ligation Enhancer
| |
| **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
| |
| **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)
| |
| **Mix:
| |
| ***5 uL DNA
| |
| ***25 uL NEBNext High Fidelity 2x PCR Master Mix
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| ***18 uL water
| |
| *** 1 uL P1 adapter primer (25 uM)
| |
| *** 1 uL P2 adapter primer (25 uM)
| |
| **PCR cycle:
| |
| ***98 degrees for 30 seconds
| |
| ***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
| |
| [[Image:Sunbirds1 PCR 2014-05-01.JPG|200px]]
| |
| 1) 1 uL DNA template, 2) 2 uL 100 bp low scale ladder, 3) 5 uL PCR product
| |
| | |
| *Bead clean up (2014-05-02)
| |
| **Use 45 uL AMPure beads (1:1)
| |
| **2 washes of 200 uL 80% EtOH
| |
| **Elute in 30 uL LowTE
| |
| | |
| *Quant DNA library using PicoGreen and run (<3 ng/uL) on BioAnalyzer DNA High Sensitivity Chip
| |
| [[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
