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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 - Skinks Plate 3 (48 wells, Reruns)== | | ==Duiker DNA Quality== |
| * 48 wells with 100ng of DNA in 20uL
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| * Digestion (2015-08-25)
| | [[Image:Duiker06292016.PNG|200px]] |
| **Mix and add to each well:
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| ***1.36 uL water
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| ***2.40 uL CutSmart Buffer
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| ***0.24 uL SbfI-HF (new)
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| **Incubation (RADIGEST on TONY):
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| ***37 degrees for 60 minutes
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| ***65 degrees for 20 minutes
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| * P1 adapter ligation (2015-08-25)
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| **Add 4 uL indexed P1 adapter (10nM) to each well, using new plate of adapters made in-house
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| **Mix and add to each well:
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| ***2.56 uL water
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| ***0.8 uL NEBuffer 4
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| ***0.32 uL ATP
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| ***0.32 uL T4 Ligase (new)
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| **Incubation (RADLIG on TONY):
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| *** 20 degrees for 60 minutes
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| *** 65 degrees for 20 minutes
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| * Clean up (2015-08-26)
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| **Take 10 uL from each well and combine to 1.7 mL tube
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| **AMPure bead clean up:
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| ***Used 430 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 - edited to get smaller average fragments (2015-08-26)
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| **BioRuptor NGS: 10 cycles of 30 seconds on, 90 seconds off, High Power
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| [[Image:Sheared08262015 01.JPG|150px]]
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| 1) 2uL 100bp low scale ladder,
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| 2) 2uL sheared DNA
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| <br>
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| *Additional 2 cycles of 30 seconds on, 90 seconds off, High Power<br>
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| [[Image:Sheared08262015 02.JPG|100px]]
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| 1) 2uL 100bp low scale ladder,
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| 2) 2uL sheared DNA
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| *Blunt End Repair (2015-08-26)
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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 (NEBENDRP on JOHN Block B):
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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 (2015-08-26)
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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 (NEBLIGAT on JOHN Block B):
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| ***20 degrees for 15 minutes
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| *Size selection - edited to select for smaller insert (2015-08-26)
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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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| ***55 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 (2015-08-26)
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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 (NEBPCR on JOHN Block B):
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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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| **Initially ran on 2% E-gel, but could not see library/template. Re-ran samples on 1% agarose gel.
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| [[Image:PCRproduct08272015.JPG|200px]] | |
| 1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product<br>
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| →Visible amplification, but size range difficult to discern.
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| *Re-run PCR to improve visibility and get more amplification (2015-08-27)
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| **Mix:
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| ***10 uL DNA
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| ***25 uL NEBNext High Fidelity 2x PCR Master Mix
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| ***13 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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| [[Image:PCRproduct08272015 2.JPG|200px]]
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| 1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product<br>
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| →Successful amplification. Clean up, quant, and Bioanalyzer will be done with other libraries.
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| *Bead clean up
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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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| <br>
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| *PicoGreen: 3.15 ng/μL
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| <br><br>
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| *Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2015-02-06)
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| [[Image:Bioanalyzer-skinks-plate1.jpg|450px]]
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| *Dilute library to 10 nM in 10 uL and send to Berkeley for sequencing (Illumina HiSeq 100 SR) (2015-02-09)
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| <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
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| __NOTOC__
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PIRE RAD Library Preps
