CTR:Notebook/PIRE/2015/08/25: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> PIRE RAD Library Preps</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> PIRE RAD Library Preps</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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***Elute in 100 uL LowTE
***Elute in 100 uL LowTE


* Sonication (2015-08-26)
* Sonication - edited to get smaller average fragments (2015-08-26)
**BioRuptor NGS: 10 cycles of 30 seconds on, 90 seconds off, High Power
**BioRuptor NGS: 10 cycles of 30 seconds on, 90 seconds off, High Power
[[Image:Skinks sheared1 2015-01-15.JPG|150px]]  
[[Image:Sheared08262015 01.JPG|150px]]  
1) 2uL 100bp low scale ladder,  
1) 2uL 100bp low scale ladder,  
2) 2uL sheared DNA
2) 2uL sheared DNA
<br>
<br>
Additional 2 cycles of 30 seconds on, 90 seconds off, High Power<br>
*Additional 2 cycles of 30 seconds on, 90 seconds off, High Power<br>
[[Image:Skinks sheared2 2015-01-15.JPG|150px]]
[[Image:Sheared08262015 02.JPG|100px]]
1) 2uL 100bp low scale ladder,
1) 2uL 100bp low scale ladder,
2) 2uL sheared DNA
2) 2uL sheared DNA
------------------------------------------------
 
*Blunt End Repair (2015-08-26)
*Blunt End Repair (2015-08-26)
**Mix:
**Mix:
Line 65: Line 65:
***20 degrees for 15 minutes
***20 degrees for 15 minutes


*Size selection (2015-08-26)
*Size selection - edited to select for smaller insert (2015-08-26)
**Add 16.5 uL water for a total of 100 uL
**Add 16.5 uL water for a total of 100 uL
**AMPure bead size selection:
**AMPure bead size selection:
***45 uL beads to remove large fragments
***55 uL beads to remove large fragments
***25 uL beads to remove small fragments
***25 uL beads to remove small fragments
***3 washes of 200 uL 80% EtOH
***3 washes of 200 uL 80% EtOH
Line 87: Line 87:
****72 degrees for 30 seconds
****72 degrees for 30 seconds
***72 degrees for 5 minutes
***72 degrees for 5 minutes
[[Image:Skinks pcr 2015-01-15.JPG|200px]]
**Initially ran on 2% E-gel, but could not see library/template. Re-ran samples on 1% agarose gel.
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product
[[Image:PCRproduct08272015.JPG|200px]]
 
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product<br>
*Bead clean up (2015-08-26)
**Use 45 uL AMPure beads (1:1)
**2 washes of 200 uL 80% EtOH
**Elute in 30 uL LowTE
 
*Quant DNA library using PicoGreen: 1.53 ng/μL. Using Qubit HS: 2.5 ng/μL. <b>→ Too low to get 10nm in 10μL.</b>
 
<br>


*Re-run of PCR using 10μL template DNA (2015-01-16)
→Visible amplification, but size range difficult to discern.
*Re-run PCR to improve visibility and get more amplification (2015-08-27)
**Mix:
**Mix:
***10 uL DNA
***10 uL DNA
Line 106: Line 99:
*** 1 uL P1 adapter primer (25 uM)
*** 1 uL P1 adapter primer (25 uM)
*** 1 uL P2 adapter primer (25 uM)
*** 1 uL P2 adapter primer (25 uM)
**PCR cycle: NEBPCR on JOHN Block B
[[Image:PCRproduct08272015 2.JPG|200px]]
[[Image:Skinks pcr 2015-01-16.JPG|200px]]
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product<br>
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product
→Successful amplification.
*PicoGreen: 3.15 ng/μL
 
<br><br>


*Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2015-02-06)
*Clean up (2015-09-02, with Mice Plate 1)
**45μL beads
**Added a second cleanup using ~28μL beads
**Ran gel to see final product
[[Image:Libraries-skinksP3 miceP1.jpg|200px]]
2μL Skink library on left


[[Image:Bioanalyzer-skinks-plate1.jpg|450px]]
*Picogreen: 1.34 ng/μL


*Dilute library to 10 nM in 10 uL and send to Berkeley for sequencing (Illumina HiSeq 100 SR) (2015-02-09)
*Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2015-09-03)
[[Image:BioAnalyzer SkinkP3 2015-09-03.jpg|550px]]


*Sent 10nM to Berkeley for sequencing (Illumina HiSeq 100 PE) (2015-09-03)


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Latest revision as of 01:05, 27 September 2017

PIRE RAD Library Preps Main project page
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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

1) 2uL 100bp low scale ladder, 2) 2uL sheared DNA

  • Additional 2 cycles of 30 seconds on, 90 seconds off, High Power

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.

1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product

→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)

1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product
→Successful amplification.

  • Clean up (2015-09-02, with Mice Plate 1)
    • 45μL beads
    • Added a second cleanup using ~28μL beads
    • Ran gel to see final product

2μL Skink library on left

  • Picogreen: 1.34 ng/μL
  • Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2015-09-03)

  • Sent 10nM to Berkeley for sequencing (Illumina HiSeq 100 PE) (2015-09-03)
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