Team:Northwestern/08 12

Notebook

Friday, August 12th

Tasks:

Jordan

  • Helped Michelle with abstract

Michelle

  • Submitted track selection, title, and abstract

Paul

  • Ran Golden Gate Reactions
  • mCherry1 GFP1-mCherry2 GFP1-GFP2-mCherry3 GFP1-GFP2-GFP3-mCherry4 GFP1-GFP2-GFP3-GFP4-mCherry5
    • 2 uL BB
    • 2 uL 10X Ligase buffer
    • 1 uL ligase
    • 0.5 uL BsaI
    • 1 uL SS
    • 1 uL mC1
    • 12.5 uL H20
    • 2 uL BB
    • 2 uL 10X Ligase buffer
    • 1 uL ligase
    • 0.5 uL BsaI
    • 1 uL SS
    • 1 uL G1
    • 1 uL mC2
    • 11.5 uL H20
    • 2 uL BB
    • 2 uL 10X Ligase buffer
    • 1 uL ligase
    • 0.5 uL BsaI
    • 1 uL SS
    • 1 uL G1
    • 1 ul G2
    • 1 uL mC3
    • 10.5 uL H20
    • 2 uL BB
    • 2 uL 10X Ligase buffer
    • 1 uL ligase
    • 0.5 uL BsaI
    • 1 uL SS
    • 1 uL G1
    • 1 ul G2
    • 1 uL G3
    • 1 uL mC4
    • 9.5 uL H20
    • 2 uL BB
    • 2 uL 10X Ligase buffer
    • 1 uL ligase
    • 0.5 uL BsaI
    • 1 uL SS
    • 1 uL G1
    • 1 ul G2
    • 1 uL G3
    • 1 ul G4
    • 1 uL mC5
    • 8.5 uL H20
  • Gibson assembly of SS+Cas9 (3:1)
    • TorA, YcdO, AmiA, FhuD, DspA, no insert control, positive control
    • 1 uL lin Cas9 BB (~50 ng)
    • 0.6 uL SS (all 10 ng/uL)
    • 8.4 uL water
    • 10 uL Gibson HiFi assembly MasterMix
    • >50°C for 45 min.
  • Autoclaved LB+agar for Cam plates

Sam

  • Diluted Paul’s saltwater to 50mM by adding 2.5 mL of the autoclaved di water
  • Made 1x TAE—still 100 mL of old 1x TAE in there with the new batch
  • Helped Michelle with abstract
  • Emailed Quentin to get the Jewett lab loading dye formula
  • Worked on human practices
  • Finally emailed Will
  • Followed up with Sarah Sutton

Sara

  • Transformed the 15 Golden Gate products and their controls with Tyler
    • Followed boot camp transformation protocol
    • Used SOC from Patrick
    • Used 200 uL SOC and plated 250 uL volume total
    • Transformed immediately, 5 uL of product per transformation
    • Also transformed a + control plasmid from NEB, 2uL
    • Excess product was stored in the freezer. Tubes were labelled as follows:
      mCherry1 mCherry2 mCherry3 mCherry4 mCherry5  
      1 2 3 4 5 TorA
      6 7 8 9 10 YcdO
      11 12 13 14 15 AmiA
      16 17 18 19 20 No ligase
      21 No insert          
  • Transformed the 6 Gibson products and their 2 controls with Tyler
    • Followed boot camp transformation protocol
    • Used SOC from Patrick
    • Used 200 uL SOC and plated 250 uL volume total
    • 2 uL of the iGEM resuspension of the tet BB
    • 3 uL of the positive gibson control
    • 3 uL of the gibson - control (no enzymes)

Shu

  • Ran a gel on RTW PCR of Cas9 products
    • Each well: 50uL sample (not exact) +10uL loading dye
    • Ladder: 4uL ladder+12uL loading dye+2uL NaCl
  • Gel extraction of linear Cas9
    • Cas9 w/ DMSO when PCR: 190mg gel is extracted
      • Concentration: 46.0ng/uL
      • 260/280: 1.88
      • 260/230: 1.41
    • Cas9 w/o DMSO when PCR: 160mg gel is extracted
      • Concentration: ~4.0ng/uL
      • 260/280: ~2.0
      • 260/230: 0.68

Tyler

  • Transformed the 6 Gibson products and their 2 controls with Sara
  • Transformed the 15 Golden Gate products and their controls with Sara

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