CTR:Notebook/PIRE/2015/01/14: Difference between revisions

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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 95: Line 95:
**Elute in 30 uL LowTE
**Elute in 30 uL LowTE


*Quant DNA library using PicoGreen and run (<3 ng/uL) on BioAnalyzer DNA High Sensitivity Chip
*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
**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)
 
----------------------
 
run (<3 ng/uL) on BioAnalyzer DNA High Sensitivity Chip
[[Image:Sunbirds1 bioanalyzer.jpg|450px]]
[[Image:Sunbirds1 bioanalyzer.jpg|450px]]


Revision as of 12:47, 16 January 2015

PIRE RAD Library Preps <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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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:
      • Use 480 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
    • 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)

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) (2015-0)
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