Team:Northwestern/09 14

Notebook

Wednesday, September 14th

Tasks:

Jordan

  • Wrote “Experiments” doc for the website
  • Transformed Gibson products
    • YcdO, NapA, ClyA, INP, AmiA, FhuD, negative control with only backbone- 3uL, positive control with 50 pg of RFP- 1 uL
    • All on Cam
    • Plated 500 uL of 50 uL cells + 950 uL SOC
    • Incubation= 1 hr 15, heat shock 30 seconds

Sam

  • Wrote Auburn Gresham scaffold
  • Looked over presentation, sent to Emma
  • Emailed Dr. DeLisa
  • Edited presentation
  • Updated OneNote
  • To do: redesign survey

Sara

  • Miniprepped Ben’s cell line, pET28a: 23 ng/uL, 260/280: 2.01, 260/230: 2.14
  • Made a glycerol stock of these cells
  • Made glycerol stock of Cas9-Dsba (1)
  • Made glycerol stock of gRNA (4)
  • Finished transforming the golden gate transformations

Tasfia

  • PCR Cleanup of INP with homology and Cas9 for restriction digest
  • Gel screen on Tet-Lrz-GFP from PCR troubleshooting
  • DpnI good Tet-Lrz-GFP PCR products (Lanes 3-5) from 09.13.16
  • PCR Cleanup of Tet-Lrz-GFP after DpnI
  • Gibson Assemblies (Shu set up the reactions, Tyler made the calculations, and Sara/I reviewed the calculations #GoTeam)
    • Cas9-NapA
      • 0.32 μL NapA with PCR homology insert
      • 0.98 μL Cas9-Lrz-SS backbone
      • 3.70 μL nuclease-free water
      • 5.00 μL Gibson Master Mix
    • Cas9-AmiA
      • 0.16 μL AmiA with PCR homology insert
      • 0.98 μL Cas9-Lrz-SS backbone
      • 3.86 μL nuclease-free water
      • 5.00 μL Gibson Master Mix
    • Cas9-YcdO
      • 0.16 μL YcdO with PCR homology insert
      • 0.98 μL Cas9-Lrz-SS backbone
      • 3.86 μL nuclease-free water
      • 5.00 μL Gibson Master Mix
    • Cas9-FhuD
      • 0.20 μL FhuD with PCR homoloy insert
      • 0.98 μL Cas9-Lrz-SS backbone
      • 3.82 μL nuclease-free water
      • 5.00 μL Gibson Master Mix
    • Cas9-ClyA
      • 1.63 μL ClyA gBlock insert
      • 0.98 μL Cas9-Lrz-ClyA backbone
      • 2.39 μL nuclease-free water
      • 5.00 μL Gibson Master Mix
    • Cas9-INP
      • 0.65 μL diluted INP (1:9 DNA-to-water dilution) with PCR homology insert
      • 0.98 μL Cas9-Lrz-SS backbone
      • 3.37 μL nuclease-free water
      • 5.00 μL Gibson Master Mix
    • Negative Control
      • 0.98 μL Cas9-Lrz-ClyA backbone
      • 4.02 μL nuclease-free water
      • 5.00 μL Gibson Master Mix
    • Reactions are running for an hour at 50°C

Tyler

  • Reviewed sequencing reactions
    • For Dsba 2,4,7,8,9, A, and B have suboptimal sequencing results, but still look to be usable
      • USE 1,3,5,6,10
    • For gRNA, gRNA 6 and 7 had suboptimal results and were thrown away
      • USE 1,2,3,4,5,8,9,10

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