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

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==RAD Library Prep - Olive Sunbirds Plate 1==
==RAD Library Prep - Olive Sunbirds Plate 1==
* 2014-04-30: Digested, P1 adapter ligated, Cleaned
* 96 well plate of 50ng of DNA 10uL
* 2014-05-01: Sonicated
 
* Digestion (2014-04-30)
**Mix and add to each well:
***0.68 uL water
***1.20 uL NEBuffer 4
***0.12 uL SbfI-HF
**Incubation:
***37 degrees for 60 minutes
***65 degrees for 20 minutes
 
* P1 adapter ligation (2014-04-30)
**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:
*** 20 degrees for 60 minutes
*** 65 degrees for 20 minutes
 
* Clean up (2014-04-30)
**Take 5 uL frm each well and combine to 1.7 mL tube
**AMPure bead clean up:
***Use 480 uL beads to DNA (1:1)
***2 washes of 800 uL 80% EtOH
***Elute in 100 uL LowTE
 
* Sonication (2014-05-01)
**BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power
[[Image:Sunbird1 sheared 2014-05-01.JPG|200px]]  
[[Image:Sunbird1 sheared 2014-05-01.JPG|200px]]  
1) 1uL pre-sheared DNA,  
1) 1uL pre-sheared DNA,  
2) 2uL 100bp low scale ladder,  
2) 2uL 100bp low scale ladder,  
3) 3uL sheared DNA
3) 3uL sheared DNA
*Blunt End Repair (2014-05-01)
**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:
***20 degrees for 30 minutes
***65 degrees for 30 minutes
*P2 adapter ligation (2014-05-01)
**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:
***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




Revision as of 13:50, 1 May 2014

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

RAD Library Prep - Olive Sunbirds Plate 1

  • 96 well plate of 50ng of DNA 10uL
  • Digestion (2014-04-30)
    • Mix and add to each well:
      • 0.68 uL water
      • 1.20 uL NEBuffer 4
      • 0.12 uL SbfI-HF
    • Incubation:
      • 37 degrees for 60 minutes
      • 65 degrees for 20 minutes
  • P1 adapter ligation (2014-04-30)
    • 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:
      • 20 degrees for 60 minutes
      • 65 degrees for 20 minutes
  • Clean up (2014-04-30)
    • Take 5 uL frm each well and combine to 1.7 mL tube
    • AMPure bead clean up:
      • Use 480 uL beads to DNA (1:1)
      • 2 washes of 800 uL 80% EtOH
      • Elute in 100 uL LowTE
  • Sonication (2014-05-01)
    • BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power

1) 1uL pre-sheared DNA, 2) 2uL 100bp low scale ladder, 3) 3uL sheared DNA

  • Blunt End Repair (2014-05-01)
    • 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:
      • 20 degrees for 30 minutes
      • 65 degrees for 30 minutes
  • P2 adapter ligation (2014-05-01)
    • 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:
      • 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
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