CTR:Notebook/PIRE/Entry Base

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(RAD Library Prep - Skinks 1)
 
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==Entry title==
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==RAD Library Prep - Skinks Plate 1==
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* Insert content here...
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* 96 well plate of 50ng of DNA 10uL
 +
 
 +
* Digestion (2015-01-14)
 +
**Mix and add to each well:
 +
***0.68 uL water
 +
***1.20 uL NEBuffer 4
 +
***0.12 uL SbfI-HF
 +
**Incubation (RADIGEST on TONY):
 +
***37 degrees for 60 minutes
 +
***65 degrees for 20 minutes
 +
 
 +
* P1 adapter ligation (2015-01-14)
 +
**Add 2 uL indexed P1 adapter (10nM) to each well, using new plate of adapters made in-house
 +
**Mix and add to each well:
 +
***1.28 uL water
 +
***0.40 uL NEBuffer 4
 +
***0.16 uL ATP
 +
***0.16 uL T4 Ligase
 +
**Incubation (RADLIG on TONY):
 +
*** 20 degrees for 60 minutes
 +
*** 65 degrees for 20 minutes
 +
 
 +
* Clean up (2015-01-15)
 +
**Take 5 uL from each well and combine to 1.7 mL tube
 +
**AMPure bead clean up:
 +
***Used 440 uL beads to DNA (1:1)
 +
***2 washes of 800 uL 80% EtOH
 +
***Elute in 100 uL LowTE
 +
 
 +
* Sonication (2015-01-15)
 +
**BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power
 +
[[Image:Skinks sheared1 2015-01-15.JPG|150px]]
 +
1) 2uL 100bp low scale ladder,
 +
2) 2uL sheared DNA
 +
<br>
 +
Additional 3 cycles of 15 seconds on, 90 seconds off, High Power<br>
 +
[[Image:Skinks sheared2 2015-01-15.JPG|150px]]
 +
1) 2uL 100bp low scale ladder,
 +
2) 2uL sheared DNA
 +
 
 +
*Blunt End Repair (2015-01-15)
 +
**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 (2015-01-15)
 +
**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 (2015-01-15)
 +
**Add 16.5 uL water for a total of 100 uL
 +
**AMPure bead size selection:
 +
***45 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 (2015-01-15)
 +
**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
 +
[[Image:Skinks pcr 2015-01-15.JPG|200px]]
 +
1) 2 uL 100 bp low scale ladder, 2) 1 uL DNA template, 3) 5 uL PCR product
 +
 
 +
*Bead clean up (2015-01-16)
 +
**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)
 +
**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)
 +
**PCR cycle: NEBPCR on JOHN Block B
 +
[[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
 +
*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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<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
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|}
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__NOTOC__
__NOTOC__

Current revision

PIRE RAD Library Preps Main project page

RAD Library Prep - Skinks Plate 1

  • 96 well plate of 50ng of DNA 10uL
  • Digestion (2015-01-14)
    • Mix and add to each well:
      • 0.68 uL water
      • 1.20 uL NEBuffer 4
      • 0.12 uL SbfI-HF
    • Incubation (RADIGEST on TONY):
      • 37 degrees for 60 minutes
      • 65 degrees for 20 minutes
  • P1 adapter ligation (2015-01-14)
    • Add 2 uL indexed P1 adapter (10nM) to each well, using new plate of adapters made in-house
    • Mix and add to each well:
      • 1.28 uL water
      • 0.40 uL NEBuffer 4
      • 0.16 uL ATP
      • 0.16 uL T4 Ligase
    • Incubation (RADLIG on TONY):
      • 20 degrees for 60 minutes
      • 65 degrees for 20 minutes
  • Clean up (2015-01-15)
    • Take 5 uL from each well and combine to 1.7 mL tube
    • AMPure bead clean up:
      • Used 440 uL beads to DNA (1:1)
      • 2 washes of 800 uL 80% EtOH
      • Elute in 100 uL LowTE
  • Sonication (2015-01-15)
    • BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power

1) 2uL 100bp low scale ladder, 2) 2uL sheared DNA
Additional 3 cycles of 15 seconds on, 90 seconds off, High Power
1) 2uL 100bp low scale ladder, 2) 2uL sheared DNA

  • Blunt End Repair (2015-01-15)
    • 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 (2015-01-15)
    • 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 (2015-01-15)
    • Add 16.5 uL water for a total of 100 uL
    • AMPure bead size selection:
      • 45 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 (2015-01-15)
    • 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

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

  • Bead clean up (2015-01-16)
    • 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. → Too low to get 10nm in 10μL.


  • Re-run of PCR using 10μL template DNA (2015-01-16)
    • 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)
    • PCR cycle: NEBPCR on JOHN Block B

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

  • 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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