Duiker DNA Quality:
←Older revision
Revision as of 23:19, 29 June 2016
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RAD Library Prep - Skinks Plate 3 (48 wells, Reruns)
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Duiker DNA Quality
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* 48 wells with 100ng of DNA in 20uL
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* Digestion (2015-08-25)
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[[Image:
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**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:
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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]]
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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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→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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