CTR:Notebook/PIRE/2015/08/31: Difference between revisions

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*** 65 degrees for 20 minutes
*** 65 degrees for 20 minutes


* Clean up (2015-08-26)
* Clean up (2015-09-01)
**Take 10 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:
Line 36: Line 36:
***Elute in 100 uL LowTE
***Elute in 100 uL LowTE


* Sonication - edited to get smaller average fragments (2015-08-26)
* Sonication - edited to get smaller average fragments (2015-09-01)
**BioRuptor NGS: 12 cycles of 30 seconds on, 90 seconds off, High Power
**BioRuptor NGS: 12 cycles of 30 seconds on, 90 seconds off, High Power
[[Image:Sheared08262015 01.JPG|150px]]  
[[Image:Sheared08262015 01.JPG|150px]]  
Line 42: Line 42:
2) 2uL sheared DNA
2) 2uL sheared DNA


*Blunt End Repair (2015-08-26)
*Blunt End Repair (2015-09-01)
**Mix:
**Mix:
***50.0 uL fragmented DNA
***50.0 uL fragmented DNA
Line 52: Line 52:
***65 degrees for 30 minutes
***65 degrees for 30 minutes


*P2 adapter ligation (2015-08-26)
*P2 adapter ligation (2015-09-01)
**Add to mix:
**Add to mix:
***15.0 uL Blunt/TA Ligase Master Mix
***15.0 uL Blunt/TA Ligase Master Mix
Line 60: Line 60:
***20 degrees for 15 minutes
***20 degrees for 15 minutes


*Size selection - edited to select for smaller insert (2015-08-26)
*Size selection - edited to select for smaller insert (2015-09-01)
**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:
Line 68: Line 68:
***Elute in 20 uL LowTE
***Elute in 20 uL LowTE


*PCR amplification (2015-08-26)
*PCR amplification (2015-09-02)
**Mix:
**Mix:
***5 uL DNA
***5 uL DNA

Revision as of 09:27, 31 August 2015

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RAD Library Prep - 48-sample libraries for Butterflies, Frogs, Mice, Skinks

  • 48 wells with 100ng of DNA in 20uL: Butterflies Plate 1 + Frogs Plate 1, Mice Plate 1 + Skinks Plate 4
  • Digestion (2015-08-31)
    • Mix and add to each well:
      • 1.36 uL water
      • 2.40 uL CutSmart Buffer
      • 0.24 uL SbfI-HF (new)
    • Incubation (B+F: RADIGEST on TONY; M+S: RADIGEST on SORK):
      • 37 degrees for 60 minutes
      • 65 degrees for 20 minutes
  • P1 adapter ligation (2015-08-31)
    • 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 (B+F: RADIGEST on TONY; M+S: RADIGEST on SORK):
      • 20 degrees for 60 minutes
      • 65 degrees for 20 minutes
  • Clean up (2015-09-01)
    • 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 (2015-09-01)
    • BioRuptor NGS: 12 cycles of 30 seconds on, 90 seconds off, High Power

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

  • Blunt End Repair (2015-09-01)
    • 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 (2015-09-01)
    • 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 (2015-09-01)
    • 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 (2015-09-02)
    • 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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