Autocreate 2016/06/29 Entry for CTR:Notebook/PIRE
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> PIRE RAD Library Preps</span>
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==RAD Library Prep - Skinks Plate 3 (48 wells, Reruns)==
* 48 wells with 100ng of DNA in 20uL
* Digestion (2015-08-25)
**Mix and add to each well:
***1.36 uL water
***2.40 uL CutSmart Buffer
***0.24 uL SbfI-HF (new)
**Incubation (RADIGEST on TONY):
***37 degrees for 60 minutes
***65 degrees for 20 minutes
* P1 adapter ligation (2015-08-25)
**Add 4 uL indexed P1 adapter (10nM) to each well, using new plate of adapters made in-house
**Mix and add to each well:
***2.56 uL water
***0.8 uL NEBuffer 4
***0.32 uL ATP
***0.32 uL T4 Ligase (new)
**Incubation (RADLIG on TONY):
*** 20 degrees for 60 minutes
*** 65 degrees for 20 minutes
* Clean up (2015-08-26)
**Take 10 uL from each well and combine to 1.7 mL tube
**AMPure bead clean up:
***Used 430 uL beads to DNA (1:1)
***2 washes of 800 uL 80% EtOH
***Elute in 100 uL LowTE
* Sonication - edited to get smaller average fragments (2015-08-26)
**BioRuptor NGS: 10 cycles of 30 seconds on, 90 seconds off, High Power
[[Image:Sheared08262015 01.JPG|150px]]
1) 2uL 100bp low scale ladder,
2) 2uL sheared DNA
<br>
*Additional 2 cycles of 30 seconds on, 90 seconds off, High Power<br>
[[Image:Sheared08262015 02.JPG|100px]]
1) 2uL 100bp low scale ladder,
2) 2uL sheared DNA
*Blunt End Repair (2015-08-26)
**Mix:
***50.0 uL fragmented DNA
***5.5 uL water
***3.0 uL End Prep Enzyme Mix
***6.5 uL End Repair Reaction Buffer
**Incubation (NEBENDRP on JOHN Block B):
***20 degrees for 30 minutes
***65 degrees for 30 minutes
*P2 adapter ligation (2015-08-26)
**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 (NEBLIGAT on JOHN Block B):
***20 degrees for 15 minutes
*Size selection - edited to select for smaller insert (2015-08-26)
**Add 16.5 uL water for a total of 100 uL
**AMPure bead size selection:
***55 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 (2015-08-26)
**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 (NEBPCR 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
**Initially ran on 2% E-gel, but could not see library/template. Re-ran samples on 1% agarose gel.
[[Image:PCRproduct08272015.JPG|200px]]
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product<br>
→Visible amplification, but size range difficult to discern.
*Re-run PCR to improve visibility and get more amplification (2015-08-27)
**Mix:
***10 uL DNA
***25 uL NEBNext High Fidelity 2x PCR Master Mix
***13 uL water
*** 1 uL P1 adapter primer (25 uM)
*** 1 uL P2 adapter primer (25 uM)
[[Image:PCRproduct08272015 2.JPG|200px]]
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product<br>
→Successful amplification. Clean up, quant, and Bioanalyzer will be done with other libraries.
*Bead clean up
**Use 45 uL AMPure beads (1:1)
**2 washes of 200 uL 80% EtOH
**Elute in 30 uL LowTE
<br>
*PicoGreen: 3.15 ng/μL
<br><br>
*Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2015-02-06)
[[Image:Bioanalyzer-skinks-plate1.jpg|450px]]
*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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