CTR:Notebook/PIRE/2015/09/09

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RAD Library Prep - 48 sample libraries for Butterflies, Frogs, Skinks P4, Skinks P5

  • 48 wells with 100ng of DNA in 20uL: Butterflies Plate 1 (re-prep) + Frogs Plate 1 (re-prep), Skinks Plate 4 (re-prep) + Skinks Plate 5
  • Digestion (2015-09-08)
    • 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; Skinks: RADIGEST on SORK):
      • 37 degrees for 60 minutes
      • 65 degrees for 20 minutes
  • P1 adapter ligation (2015-09-08)
    • 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 CutSmart Buffer
      • 0.32 uL ATP
      • 0.32 uL T4 Ligase (new)
    • Incubation (B+F: RADLIG on TONY; Skinks: RADLIG on SORK):
      • 20 degrees for 60 minutes
      • 65 degrees for 20 minutes
  • Clean up (2015-09-09)
    • Take 10 uL from each well and combine to 1.7 mL tube
    • AMPure bead clean up:
      • Used 430-440 uL beads to DNA (1:1)
      • 2 washes of 800 uL 80% EtOH
      • Elute in 100 uL LowTE
  • Sonication (2015-09-09)
    • BioRuptor NGS: 12 cycles of 30 seconds on, 90 seconds off, High Power

Sheared 09092015.jpg
1) 2μL sheared Butterfly DNA
3) 2μL sheared Frog DNA
5) 2μL sheared Skink P4 DNA
7) 2μL sheared Skink P5 DNA

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

PCR 09092015.jpg.png

  1. 1μL Butterfly template
  2. 5μL Butterfly PCR product
  3. 1μL Frog template
  4. 5μL Frog PCR product
  5. 2μL 100bp low scale ladder
  6. 1μL Skink P4 template
  7. 5μL Skink P4 product
  8. 1μL Skink P5 template
  9. 5μL Skink P5 PCR product

→Successful amplification for Skinks P4 (PCR product is visibly brighter. Butterfly, Frog and Skink P5 shows some amplification (~same brightness as template).

  • Re-run Butterfly, Frog, and Skink P5 PCR (2015-09-10)
    • 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)

PCR2 09102015.jpg.png

  1. 1μL Butterfly template
  2. 5μL Butterfly PCR product
  3. 2μL 100bp low scale ladder
  4. 1μL Frog template
  5. 5μL Frog PCR product
  6. 1μL Skink P5 template
  7. 5μL Skink P5 PCR product

→ Clear amplification of Butterflies and Frogs. Skinks P5 shows no amplication, same brightness as template despite 2X increase of starting material in PCR reaction.

  • Bead clean up of Butterflies, Frogs, and Skinks P4 (2015-09-10)
    • Used 45μL AMPure beads (1:1)
    • Added second clean up using ~28μL beads (1:1)
  • PicoGreen:
    • Butterfly: -0.305 ng/μL
    • Frog: 1.89 ng/μL
    • Skink P4: 1.95 ng/μL

→ Butterfly concentration is too low. Repeat library prep from the blunt end repair step, using 40 ul of the leftover sheared DNA and added 15.5 ul of water to try to get more DNA (2015-09-10)

  • Re-prep for sheared Butterfly DNA
  • Blunt End Repair (2015-09-10)
    • 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 (2015-09-10)
    • 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 (2015-09-10)
    • 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 (2015-09-11)
    • 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):
      • 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

Butterfly PCR3.png

  1. 2μL 100bp low scale ladder
  2. 1μL Butterfly template
  3. 5μL Butterfly PCR product

→Successful amplification.

  • Bead clean up of Butterflies, Frogs, and Skinks P4 (2015-09-11)
    • Used 45μL AMPure beads (1:1)
    • Added second clean up using ~28μL beads (1:1)
  • Combined with 1st library prep
  • PicoGreen: 1.41 ng/μL


  • Run 3μL on BioAnalyzer DNA High Sensitivity Chip (2015-09-16)
    • Butterfly Plate 1:

ButterflyP1.jpg

  • Frog Plate 1:

FrogsP1.png

  • Skink Plate 4:

SkinksP4.jpg

  • Sent 10nM to Berkeley for sequencing (Illumina HiSeq 100 PE) (2015-09-17)