CTR:Notebook/PIRE/Entry Base: Difference between revisions

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==RAD Library Prep - Olive Sunbirds Plate 1==
==RAD Library Prep - Skinks Plate 3 (48 wells, Reruns)==
* 96 well plate of 50ng of DNA 10uL
* 48 wells with 100ng of DNA in 20uL


* Digestion (2014-04-30)
* Digestion (2015-08-25)
**Mix and add to each well:
**Mix and add to each well:
***0.68 uL water
***1.36 uL water
***1.20 uL NEBuffer 4
***2.40 uL CutSmart Buffer
***0.12 uL SbfI-HF
***0.24 uL SbfI-HF (new)
**Incubation:
**Incubation (RADIGEST on TONY):
***37 degrees for 60 minutes
***37 degrees for 60 minutes
***65 degrees for 20 minutes
***65 degrees for 20 minutes


* P1 adapter ligation (2014-04-30)
* P1 adapter ligation (2015-08-25)
**Add 2 uL indexed P1 adapter (10nM) to each well
**Add 4 uL indexed P1 adapter (10nM) to each well, using new plate of adapters made in-house
**Mix and add to each well:
**Mix and add to each well:
***1.28 uL water
***2.56 uL water
***0.40 uL NEBuffer 4
***0.8 uL NEBuffer 4
***0.16 uL ATP
***0.32 uL ATP
***0.16 uL T4 Ligase
***0.32 uL T4 Ligase (new)
**Incubation:
**Incubation (RADLIG on TONY):
*** 20 degrees for 60 minutes
*** 20 degrees for 60 minutes
*** 65 degrees for 20 minutes
*** 65 degrees for 20 minutes


* Clean up (2014-04-30)
* Clean up (2015-08-26)
**Take 5 uL from each well and combine to 1.7 mL tube
**Take 10 uL from each well and combine to 1.7 mL tube
**AMPure bead clean up:
**AMPure bead clean up:
***Use 480 uL beads to DNA (1:1)
***Used 430 uL beads to DNA (1:1)
***2 washes of 800 uL 80% EtOH
***2 washes of 800 uL 80% EtOH
***Elute in 100 uL LowTE
***Elute in 100 uL LowTE


* Sonication (2014-05-01)
* Sonication - edited to get smaller average fragments (2015-08-26)
**BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power
**BioRuptor NGS: 10 cycles of 30 seconds on, 90 seconds off, High Power
[[Image:Sunbird1 sheared 2014-05-01.JPG|200px]]  
[[Image:Sheared08262015 01.JPG|150px]]  
1) 1uL pre-sheared DNA,  
1) 2uL 100bp low scale ladder,
2) 2uL 100bp low scale ladder,  
2) 2uL sheared DNA
3) 3uL 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 (2014-05-01)
*Blunt End Repair (2015-08-26)
**Mix:
**Mix:
***50.0 uL fragmented DNA
***50.0 uL fragmented DNA
Line 49: Line 53:
***3.0 uL End Prep Enzyme Mix
***3.0 uL End Prep Enzyme Mix
***6.5 uL End Repair Reaction Buffer
***6.5 uL End Repair Reaction Buffer
**Incubation:
**Incubation (NEBENDRP on JOHN Block B):
***20 degrees for 30 minutes
***20 degrees for 30 minutes
***65 degrees for 30 minutes
***65 degrees for 30 minutes


*P2 adapter ligation (2014-05-01)
*P2 adapter ligation (2015-08-26)
**Add to mix:
**Add to mix:
***15.0 uL Blunt/TA Ligase Master Mix
***15.0 uL Blunt/TA Ligase Master Mix
***2.5 uL P2 RAD adapter (5 uM)
***2.5 uL P2 RAD adapter (5 uM)
***1.0 uL Ligation Enhancer  
***1.0 uL Ligation Enhancer  
**Incubation:
**Incubation (NEBLIGAT on JOHN Block B):
***20 degrees for 15 minutes
***20 degrees for 15 minutes


*Size selection (2014-05-01)
*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
***Elute in 20 uL LowTE
***Elute in 20 uL LowTE


*PCR amplification (2014-05-01)
*PCR amplification (2015-08-26)
**Mix:
**Mix:
***5 uL DNA
***5 uL DNA
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*** 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:
**PCR cycle (NEBPCR on JOHN Block B):
***98 degrees for 30 seconds
***98 degrees for 30 seconds
***15 cycles of:
***15 cycles of:
Line 83: Line 87:
****72 degrees for 30 seconds
****72 degrees for 30 seconds
***72 degrees for 5 minutes
***72 degrees for 5 minutes
[[Image:Sunbirds1 PCR 2014-05-01.JPG|200px]]
**Initially ran on 2% E-gel, but could not see library/template. Re-ran samples on 1% agarose gel.
1) 1 uL DNA template, 2) 2 uL 100 bp low scale ladder, 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>
 
→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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Revision as of 09:19, 31 August 2015

PIRE RAD Library Preps <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page

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, 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


  • PicoGreen: 3.15 ng/μL



  • Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2015-02-06)

  • 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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