CTR:Notebook/PIRE/2015/02/02: Difference between revisions

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==RAD Library Prep - Olive Sunbirds Plate 1==
==RAD Library Prep - Greenbuls and Sunbirds Plate 3==
* 96 well plate of 50ng of DNA 10uL
* 96 well plate of 50ng of DNA 10uL


* Digestion (2014-04-30)
2015-01-31:
* Digestion
**Mix and add to each well:
**Mix and add to each well:
***0.68 uL water
***0.68 uL water
***1.20 uL NEBuffer 4
***1.20 uL NEBuffer 4
***0.12 uL SbfI-HF
***0.12 uL SbfI-HF
**Incubation:
**Incubation (RADIGEST: Greenbuls on SORK, Sunbirds 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
**Add 2 uL indexed P1 adapter (10nM) to each well
**Add 2 uL indexed P1 adapter (10nM) to each well
**Mix and add to each well:
**Mix and add to each well:
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***0.16 uL ATP
***0.16 uL ATP
***0.16 uL T4 Ligase
***0.16 uL T4 Ligase
**Incubation:
**Incubation (RADLIG: Greenbuls on SORK, Sunbirds 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)
2015-02-01:
* Clean up  
**Take 5 uL from each well and combine to 1.7 mL tube
**Take 5 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)
***Use 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
**BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power
**BioRuptor NGS: Left - 8 cycles of 15 seconds on, 90 seconds off, High Power. Right - Additional 6 cycles for Greenbuls, 3 cycles for Sunbirds.
[[Image:Sunbird1 sheared 2014-05-01.JPG|200px]]  
[[Image:Birdsplate3-sheared.jpg|300px]]  
1) 1uL pre-sheared DNA,  
1) 2μL sheared Greenbul DNA,  
2) 2uL 100bp low scale ladder,  
2) 2uL 100bp low scale ladder,  
3) 3uL sheared DNA
3) 2uL sheared Sunbird DNA


*Blunt End Repair (2014-05-01)
2015-02-02:
*Blunt End Repair
**Mix:
**Mix:
***50.0 uL fragmented DNA
***50.0 uL fragmented DNA
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***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
**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
**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:
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***Elute in 20 uL LowTE
***Elute in 20 uL LowTE


*PCR amplification (2014-05-01)
*PCR amplification
**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:
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***72 degrees for 5 minutes
***72 degrees for 5 minutes
[[Image:Sunbirds1 PCR 2014-05-01.JPG|200px]]
[[Image:Sunbirds1 PCR 2014-05-01.JPG|200px]]
1) 1 uL DNA template, 2) 2 uL 100 bp low scale ladder, 3) 5 uL PCR product
1) 1μL Greenbul DNA template, 2) 5μL Greenbul PCR product, 3) 2 uL 100 bp low scale ladder, 4) 1μL Sunbird DNA template, 5) 5μL Sunbird PCR product


*Bead clean up (2014-05-02)
2015-02-03:
*Bead clean up
**Use 45 uL AMPure beads (1:1)
**Use 45 uL AMPure beads (1:1)
**2 washes of 200 uL 80% EtOH
**2 washes of 200 uL 80% EtOH

Revision as of 17:26, 2 February 2015

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RAD Library Prep - Greenbuls and Sunbirds Plate 3

  • 96 well plate of 50ng of DNA 10uL

2015-01-31:

  • Digestion
    • Mix and add to each well:
      • 0.68 uL water
      • 1.20 uL NEBuffer 4
      • 0.12 uL SbfI-HF
    • Incubation (RADIGEST: Greenbuls on SORK, Sunbirds on TONY)
      • 37 degrees for 60 minutes
      • 65 degrees for 20 minutes
  • P1 adapter ligation
    • Add 2 uL indexed P1 adapter (10nM) to each well
    • 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: Greenbuls on SORK, Sunbirds on TONY)
      • 20 degrees for 60 minutes
      • 65 degrees for 20 minutes

2015-02-01:

  • Clean up
    • Take 5 uL from each well and combine to 1.7 mL tube
    • AMPure bead clean up:
      • Use 430 uL beads to DNA (1:1)
      • 2 washes of 800 uL 80% EtOH
      • Elute in 100 uL LowTE
  • Sonication
    • BioRuptor NGS: Left - 8 cycles of 15 seconds on, 90 seconds off, High Power. Right - Additional 6 cycles for Greenbuls, 3 cycles for Sunbirds.

1) 2μL sheared Greenbul DNA, 2) 2uL 100bp low scale ladder, 3) 2uL sheared Sunbird DNA

2015-02-02:

  • Blunt End Repair
    • 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
    • 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
    • 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
    • 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) 1μL Greenbul DNA template, 2) 5μL Greenbul PCR product, 3) 2 uL 100 bp low scale ladder, 4) 1μL Sunbird DNA template, 5) 5μL Sunbird PCR product

2015-02-03:

  • Bead clean up
    • Use 45 uL AMPure beads (1:1)
    • 2 washes of 200 uL 80% EtOH
    • Elute in 30 uL LowTE
  • Quant DNA library using PicoGreen and run (<3 ng/uL) on BioAnalyzer DNA High Sensitivity Chip

  • Dilute library to 10 nM in 10 uL and send to Berkeley for sequencing (Illumina HiSeq 100 SR) (2014-05-05)
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